A comparison of the responses of dispersed steroidogenic cells derived from embryonic adrenal tissue from the domestic chicken (Gallus domesticus), the domestic Pekin duck and the wild mallard duck (Anas platyrhynchos), and the domestic muscovy duck (Cairina moschata).

The steroidogenic responsiveness of adrenal cell suspensions prepared from domestic chicken adrenal tissue at the end of embryogenesis was compared to the responses of similar preparations derived from the wild and domesticated mallard duck (Anas platyrhynchos), and the domesticated muscovy duck (Cairina moschata). In all cases, the masses of corticosterone (B), aldosterone (Aldo), and deoxycorticosterone (DOC) released from cells incubated in medium containing 1-24 ACTH exceeded the estimated hormone content of the freshly dispersed cells; the induced rates of corticosteroid release were, therefore, presumed to reflect de novo hormone synthesis. When chicken cells were incubated in medium containing 1-24 ACTH, there were progressive, dose-dependent increases in B and DOC synthesis over a range of concentrations spanning two orders of magnitude; only small, non-dose-related, albeit significant, increases in Aldo release were observed. The 1-24 ACTH-induced increases in B and Aldo synthesis by the mallard and Pekin duck cells exposed to the same range of concentrations were up to 40 and 60 times greater than the corresponding responses of the chicken cells. The rates of 1-24 ACTH-induced B and DOC release from muscovy duck cells were similar to those from the Pekin duck cells; compared with the mallard duck cells, however, the muscovy duck cells were less sensitive and the maximum inducible rate of B release was significantly lower. The pattern of 1-24 ACTH-induced Aldo release from the muscovy duck cells was indistinguishable from that of the mallard duck cells, although the maximum inducible increase occurred at a lower concentration. Angiotensin II (AII) induced very small, but significant increased in B, Aldo, and DOC release from the chicken cells but in a nondose-related fashion. In contrast, mallard, Pekin, and muscovy duck cells all responded in a dose-dependent manner when incubated in medium containing AII. In each instance the maximum rate of Aldo synthesis induced by AII was about one-tenth of the corresponding rate induced by 1-24 ACTH. The maximum rates of B synthesis induced by AII, however, were extremely low compared with the rates induced by 1-24 ACTH. Thus, when maximally stimulated with AII the B:Aldo output ratio ranged from 1.0 to 1.9, whereas the corresponding ratio was between 9.0 and 17.0 when the cells were maximally stimulated with 1-24 ACTH.


[Anti-Rho[D] antigen monoclonal antibodies. II. Serological characteristics of monoclonal IgG1 antibodies].

Four IgG1 monoclonal antibodies (MoAbs) to the Rh antigen D, produced by stable Epstein-Barr virus transformed B-lymphoblastoid cell lines were assessed in different serological tests. Their suitability in blood group typing was shown in antiglobulin, enzyme or albumin methods. The specificity and activity of MoAbs was tested with a panel of red cells of various Rh-phenotypes. The supernatants of all four lines showed anti-D specificity and ability to react with Du red cells. Mean level of MoAbs concentration was near 10 micrograms/ml. These human anti-D MoAbs proved to be useful D-typing diagnostic reagents.


Stimulatory effects of serotonin on maturational gonadotropin release in the Atlantic croaker, Micropogonias undulatus.

The effects of serotonin (5-HT) injection alone, and in combination with des Gly10 [D-Trp6]-luteinizing hormone-releasing hormone ethylamide (LHRHa), on plasma maturational gonadotropin (GtH) levels in the Atlantic croaker, Micropogonias undulatus, were examined. An injection of 5-HT (20 micrograms/g body wt) alone did not significantly elevate GtH levels, whereas simulatneous administration of LHRHa (20 ng/g body wt) and 5-HT elicited a greater elevation of plasma GtH levels than that induced by LHRHa alone. Pretreatment of 1-year-old fish with fluoxetine (10 micrograms/g), a 5-HT reuptake inhibitor, only slightly augmented the effect of 5-HT on GtH levels, whereas pretreatment with ketanserin (10 micrograms/g), a 5-HT receptor antagonist, completely inhibited the potentiating effect of 5-HT on the GtH response to LHRHa. Administration of LHRHa (20 ng/g) or 5-HT (20 micrograms/g) significantly elevated GtH levels in the 2- and 3-year-old croaker, but the combined treatment failed to increase GtH levels above those induced by LHRHa alone. However, with a lower dose of LHRHa (5 ng/g), the combination produced an additive effect. Serotonin (20 micrograms/ml media) alone, and in combination with LHRHa (10 ng/ml media), significantly stimulated GtH release from the pituitaries of gonadally mature 2- and 3-year-old female croaker during an 18-hr incubation in vitro, but not from pituitaries of gonadally regressed fish. The combined treatment of LHRHa with 5-HT also significantly stimulated in vitro GtH release during 6- and 12-hr pituitary incubations, whereas these treatments alone were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)


Asynchrony of changes in tissue and plasma thyroid hormones during the parr-smolt transformation of coho salmon.

The relationship between plasma thyroid hormone concentrations and the thyroid hormone concentrations in selected tissues was examined throughout the spring during the typical course of parr-smolt transformation in coho salmon (Oncorhynchus kisutch) in fresh water and also in coho salmon moved prematurely to seawater. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) were extracted from brain, liver, and muscle tissue. The T4 and T3 concentrations in the extracts and plasma were measured by radioimmunoassay. The peak in plasma T4 occurred in late April; however, the concentration of T4 in the brain and liver increased before levels of T4 in plasma increased. During the rise in plasma T4, the T4 content in muscle decreased. Plasma T3 concentrations were unchanged in March and April, but decreased in May. Transfer to seawater eliminated the late April peak in plasma T4 levels, indicating suppressed thyroid activity; however, the tissues of salmon in seawater contained more T3 than tissues of salmon in fresh water at this time. These findings indicate complex peripheral regulation of thyroidal status in this teleost and represent the first bridge between compartmental models of thyroid hormone kinetics and actual measurement of tissue pools of thyroid hormones in an ectothermic vertebrate. In summary, tissue concentrations of thyroid hormones did not echo plasma concentrations, indicating that thyroidal status cannot be inferred from plasma data alone.


[Cyclic neutropenia: a disease or a syndrome?].

Peripheral blood and bone marrow morphology, blood and bone marrow lymphocyte subpopulation composition were studied in two children with cyclic neutropenia, using flow cytofluorometry, monoclonal antibodies, colony-forming capacity of granulocytic macrophagal precursors in semi-fluid agar. The studies were conducted in varying periods of the neutropenic cycle. Differences were revealed in immunohematologic parameters and clinical course of neutropenia in the two patients. The analysis of the literature data and the authors' own observations of the patients with cyclic neutropenia permitted a suggestion on high heterogeneity of pathogenetic mechanisms of this disease.


Hormonal control of in vitro vitellogenin synthesis in Rana esculenta liver: effects of mammalian and amphibian growth hormone.

Estradiol 17-beta is known to induce hepatic synthesis and secretion of vitellogenin in all species studied and in Rana esculenta, previous experiments demonstrated the involvement of pituitary in these processes; indeed, in addition to estradiol 17-beta, homologous pituitary homogenate directly stimulated male and female liver to produce vitellogenin in tissue cultures. Therefore, the effect of ovine growth hormone (o-GH) and Rana catesbeiana growth hormone (f-GH) on hepatic vitellogenin synthesis was investigated. In the present in vitro experiments, both o-GH and f-GH positively stimulated vitellogenin synthesis, in female and male liver, in a dose-related fashion. No significant differences were found in VTG levels induced by o-GH and f-GH. The GH stimulatory effects, found during the different phases of the reproductive cycle, displayed different trends related to season and sex.


Thyroid hormone feedback regulation of the secretion of bioactive thyrotropin in the frog.

Thyrotropin-releasing hormone (TRH), ovine corticotropin-releasing hormone (oCRH) (both 268 nM), and mammalian gonadotropin-releasing hormone (mGnRH) (268 and 2680 nM) stimulated the secretion of bioactive thyrotropin (TSH) by Rana esculenta pituitaries (pars distalis) in vitro. Preincubation of the pituitaries with 50 ng/ml (64 nM) thyroxine (T4) for 6 hr suppressed the TRH- and oCRH-induced (268 nM) secretion of bioactive TSH, but did not affect the response of the pituitaries to 268 nM mGnRH. Triiodothyronine (T3) (64 nM) reduced both the TRH- and mGnRH-stimulated release of bioactive TSH; the response of TSH to TRH even decreased toward basal levels while a significant TSH response to mGnRH remained. In a separate experiment, pituitaries were preincubated for 6 hr with different equimolar doses of T3 and T4 (6.4, 32, and 64 nM); neither treatment affected the mGnRH-stimulated secretion of bioactive TSH. On the other hand, T4 suppressed the TSH response to TRH in a dose-dependent manner. The inhibitory effects of thyroid hormones on the TRH-induced release of bioactive TSH was present for at least 4 hr after their removal from the incubation medium. These results suggest that thyroid hormones exert a negative feedback control on the secretion of bioactive TSH in adult frogs by a direct action on the pars distalis. There may also be differences in thyroid hormone sensitivities of the TSH responses to mGnRH and TRH.


Effects of atrial natriuretic peptide and toad heart extract on isolated toad Bufo arenarum aortic rings.

The vascular effects of synthetic atrial natriuretic peptide (ANP) (rANP-99-126), and toad heart extract (THE) were examined on isolated toad aortic rings from the toad Bufo arenarum. ANP inhibited contraction produced by human angiotensin II (AT II), norepinephrine (NE), and arginine vasopressin (AVP) in isolated toad aortic rings. The present data show that a relaxant effect of ANP could be obtained also in the noncontracted aortic smooth muscle of toad if it had been previously challenged with AT II or NE and allowed to return to the original basal tension. Bufo arenarum THE was able to relax the AT II-induced contraction in toad aortic rings. In toad arteries contracted with 10(-6) M AT II, ANP produced a dose-dependent relaxation with a half-maximal inhibitory concentration IC50 of 1.2 x 10(-8) M. ANP was not effective in relaxing contraction induced by high K+. The vasorelaxant effect of ANP on AT II-induced contraction was significantly increased in Ca(2+)-free medium containing 3 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA-Ringer) or by pretreatment with the calcium antagonist, diltiazem (DIL). The vasorelaxant effect of ANP on basal tension after treatment with AT II was also obtained in absence of extracellular calcium (EGTA-Ringer). These results show that Bufo arenarum contains ANP-like material and that the ANP relaxant action in the toad aorta is similar to that in mammals.


Annual changes in the binding of follicle-stimulating hormone to gonads and plasma gonadotropin concentrations in Indian weaver birds inhabiting the subtropical zone.

In birds, annual changes in gonadal weight are much more pronounced in the male than in the female. To analyze the mechanism inducing such a sex difference in the gonadal responsiveness to natural environmental conditions, we measured annual changes in the binding of follicle-stimulating hormone (FSH) to the gonads and the plasma gonadotropin concentration in adult Indian weaver birds inhabiting the subtropical zone. The binding of FSH was highly specific for mammalian FSHs and located primarily in the gonad. The testicular weight and FSH binding showed marked changes during the annual breeding cycle. The testicular weight was maximal in the breeding phase (June, 1987) and minimal in the nonbreeding phase (December, 1987). FSH binding per unit testicular weight was greatest in the nonbreeding phase, while the total FSH binding per two testes was maximal in the breeding phase and minimal in the regressive phase (October, 1987). In contrast, the changes in the ovarian weight and FSH binding to the ovary were less pronounced than those in the testis. Although FSH binding per unit ovarian weight showed a peak in the nonbreeding phase, there was no significant change in the total FSH binding per ovary during the year, indicating the presence of a clear sex difference in the total FSH binding. Scatchard plot analyses of the binding suggested that the dissociation constant (Kd) ranged from 0.35 to 1.53 nM regardless of sex and season and that the changes in FSH binding were due to changes in the number of binding sites. Plasma FSH and luteinizing hormone (LH) levels markedly changed during the year in both sexes. Both FSH and LH levels were maximal in the breeding phase and minimal in the nonbreeding phase regardless of sex, although the rate of change in either hormone tended to be greater in the male than in the female.


The effects of confinement stress on circulating prolactin levels in rainbow trout (Oncorhynchus mykiss) in fresh water.

Rainbow trout were confined for 48 hr, during which time water quality either was allowed to deteriorate (resulting in elevated NH3, elevated free CO2, and reduced dissolved O2) or was maintained at preconfinement levels. Fish were removed and blood samples taken at 0, 2, 4, 8, 24, and 48 hr after the onset of confinement from both stressed (confined) and unstressed (unconfined) fish. Plasma cortisol and plasma prolactin (PRL) levels were determined using specific RIAs. Chronic confinement of rainbow trout, accompanied by a decline in water quality, resulted in significant elevation of plasma cortisol, maintained for the period of confinement. Plasma PRL levels were significantly lower in stressed fish, by up to 60% relative to control fish, during the first 24 hr of confinement. The stress of confinement alone, in the absence of deterioration in water quality, produced similar results, with the change in prolactin levels being less rapid but more prolonged under these conditions.


Hormonal control of polymorphic and sexually dimorphic coloration in the lizard Sceloporus undulatus erythrocheilus.

This study investigated the influence of sex steroids on the expression of seasonally labile and sexually dimorphic coloration in the red-lipped plateau lizard. The responses of ventral blue and polymorphic facial coloration to exogenous steroid administration were assessed. Though facial color is usually dimorphic in nature, males and females did not differ in the degree to which they expressed facial coloration following administration of testosterone (T). Both sexes developed significantly more ventral blue coloration than controls, but a fundamental dimorphism in intensity, favoring males, was maintained throughout the experiment. These results indicate that facial coloration is under activational influence of T. The ventral blue coloration is probably organized at sexual maturation, thus manifesting the more vivid expression in males. Males of this subspecies are polymorphic for facial coloration. Administration of T did not cause any lizards to change hue, whether their color morph was yellow or orange or lacked the facial pigments altogether. The difference among color morphs is probably not controlled by differences in circulating steroid concentrations and is hypothesized to be under genetic control.


Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria.

Structural and regulatory genes encoding enzymes and proteins of the reductive pentose phosphate pathway have been isolated from a number of bacteria recently. In the phototroph Rhodobacter sphaeroides, and in two chemoautotrophic bacteria, Alcaligenes eutrophus and Xanthobacter flavus, these genes have been found in distinct operons. However, in these three organisms and in other bacteria where certain of these genes have been discovered, a uniform nomenclature to designate these genes has been lacking. This report represents an effort to provide uniformity to the designation of these genes from all bacteria.


Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates.

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.


In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.


Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen.

The distribution of two xylanase genes was examined by Southern hybridization among 26 strains of the rumen anaerobic bacterium Prevotella (Bacteroides) ruminicola. Hybridization with a xylanase/endoglucanase gene from the type strain 23 was found in six strains while hybridization with a xylanase gene from strain D31d was found in 14 strains. Sequences related to both genes were present, on different restriction fragments, in six strains, whereas no hybridization to either gene was detected in five other strains capable of hydrolysing xylan, or in seven strains that showed little or no xylanase activity. Zymogram analyses of seven xylanolytic strains of P. ruminicola demonstrated interstrain variation in the apparent molecular masses of the major xylanases and carboxymethylcellulases that could be renatured following SDS polyacrylamide gel electrophoresis.


Potassium ions and changes in bacterial DNA supercoiling under osmotic stress.

Escherichia coli transiently increases both the [ATP]/[ADP] ratio and the negative supercoiling of plasmid DNA when it is shifted to high osmolarity. Here we report that a mutant lacking all saturable K+ transport systems increases the negative supercoiling of the plasmid DNA under upshock but cannot further relax DNA. The mutant dnaK756 behaves like the K+ transport mutant.


Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.


Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans.

Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OTCase gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.


Multiple transformation of Saccharomyces cerevisiae by protoplast fusion.

A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.


Glycosaminoglycans inhibit Candida albicans adherence to extracellular matrix proteins.

The ability of Candida albicans to adhere to subendothelial extracellular matrix (ECM) may be important in the pathogenesis of disseminated candidiasis. ECM proteins, such as fibronectin, laminin, and types I and IV collagen bind C. albicans avidly. These proteins all possess heparin-binding domains. The influence of the glycosaminoglycans (GAGS) including heparin, heparan sulfate and dextran sulfate on C. albicans adherence to subendothelial ECM and ECM proteins was studied. It was demonstrated that the GAGS inhibited C. albicans adherence to ECM and ECM proteins. This possibly occurred by the GAGS binding to the ECM proteins and, in so doing, masking a preferred ligand for C. albicans adherence.


Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil.

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.


Cloning of DNA from a Rhodococcus strain conferring the ability to decolorize sulfonated azo dyes.

Azo dyes are recalcitrant pollutants. Two sulfonated azo dyes, Orange II and Amido black, are effectively decolorized by certain nocardioform strains of the genus Rhodococcus. A mutant of one of these strains was isolated which had lost azo-dye decolorizing ability and the strain was used to clone DNA conferring this ability, by screening a BclI library constructed from DNA of a decolorizing strain. The relevant genetic information was located on a 6.3-kb fragment of DNA.


Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D.

Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M(r) 41,000 and M(r) 40,000 as the phaECv and phaCCv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression of poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein (M(r) 17,000) exhibited no homology to other proteins. Lysozyme, which was added during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.


Glucoamylase P gene of Hormoconis resinae: molecular cloning, sequencing and introduction into Trichoderma reesei.

The glucoamylase P gene of the fungus Hormoconis resinae has been cloned and sequenced from a genomic library. The gene consists of a 2153-bp protein coding region including three introns. The usual number of introns in cloned fungal glucoamylase genes has been four and in some cases five. Two of the glucoamylase P gene introns contain a sequence resembling the consensus sequence found near the 3' splice site in the introns of the fungus Trichoderma reesei cellobiohydrolase 1 (cbh1) gene. The H. resinae glucoamylase P gene, under its own promoter, was introduced into T. reesei, but no expression could be detected.


Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible locations for its origin of replication.

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.


Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains.

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.


Construction of a shuttle lacZ alpha-based Escherichia coli-actinomycetes vector containing the phage JHJ-3 replicon.

Using the broad replicating range JHJ-3 phage replicon, a shuttle vector for Escherichia coli and actinomycetes has been constructed. The vector, pOJ31, bears the lacZ alpha fragment allowing a blue/white gene cloning system. pOJ31 also contains a polylinker of 15 unique cloning sites and the phage T7 promoter. The vector has been used to stably express the mel gene from plasmid pIJ702 in Streptomyces lividans.


Reversible expression of flagella in Campylobacter spp.

The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C. jejuni, C. coli, C. lari (thermophilic campylobacters), C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis were investigated. The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied. When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C. fetus and C. hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively. Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C. fetus strains. No reversion from aflagellate to flagellate phenotype could be detected in C. hyointestinalis strains. It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.


Molecular cloning and expression of the xylanase gene from Chainia in Escherichia coli.

A complete genomic library of Chainia was constructed in coliphage lambda vector gt10 and was screened for the xylanase gene using an 18-mer mixed oligonucleotide probe corresponding to a six-amino acid sequence of low molecular mass Chainia xylanase. Inserts from 11 putative clones, showing hybridization with the oligonucleotide probe at medium stringency, were subcloned in pUC8 and screened for xylanase gene expression using anti-xylanase antibodies. The restriction map of the insert (1.4 kb) from one of the four immunopositive clones (PVX8) showing detectable xylanase activity was constructed. The xylanase activity of PVX8 was not induced by IPTG or xylan. Reorientation of the insert by directional cloning into pUC9 had no effect on the xylanase activity suggesting that an indigenous promoter from Chainia is responsible for the xylanase activity.


The putative sigma factor KatF (RpoS) is required for the transcription of the Salmonella typhimurium virulence gene spvB in Escherichia coli.

The virulence of Salmonella typhimurium for mice is dependent on a plasmid-borne gene cluster termed spv. We previously determined that both S. typhimurium and Escherichia coli bacteria grown in a rich medium preferentially express the spv genes during the stationary phase of growth. In this study we evaluated the role of KatF, a putative sigma factor for starvation- and stationary phase-induced genes, in the expression of the spvB gene. The transcription of spvB in E. coli was compared in katF and wild-type backgrounds, using cloned spvB-lacZ and spvB-cat fusions. Expression of spvB was found to be greatly affected in katF mutants. Complementation experiments performed with the cloned katF gene confirmed that KatF is required for the expression of the S. typhimurium virulence gene spvB in E. coli.


The nature and site of biocide-induced sublethal injury in Bacillus subtilis spores.

Spores of Bacillus subtilis NCTC 8236 exposed at 22 degrees C to test biocides (alkaline glutaraldehyde, an iodophor, Lugol's solution, sodium hypochlorite and sodium dichloroisocyanurate) demonstrated varying degrees of injury to stressing agents (sodium hydroxide, sodium lauryl sulphate, polymyxin B sulphate or cetylpyridinium chloride) incorporated into a recovery agar medium. This injury to stressing agents was expressed mainly during outgrowth.


Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.


Isolation and partial characterization of SSI-like protease inhibitors from Streptomyces.

We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.


The PurR binding site in the glyA promoter region of Escherichia coli.

Site-directed mutagenesis was used to change the PurR binding site in the control region of a glyA-lac gene fusion. Mutations that changed the PurR binding sequence away from the consensus sequence reduced PurR binding, which correlated with reduced purine-mediated repression. Mutations that changed the binding sequence toward the consensus sequence had no significant effect on either PurR binding or purine-mediated repression. Hypoxanthine and guanine, co-repressors for PurR-mediated regulation of the pur regulon, increased binding of PurR to glyA operator DNA.


Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group.

Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes. The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis. The presence of the hydrolase was also confirmed by enzyme assay. In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P. diminuta and P. vesicularis. An absence of cytosine deaminase activity was found when assaying extracts of the two type strains. Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P. vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P. diminuta growth on dihydrothymine as a nitrogen source.


Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing.

Conventional methods of detecting polymerase chain reaction (PCR) products require equipment and expertise which may not be available in diagnostic bacteriology laboratories, especially in developing countries. To this end we have examined other methods of product detection, including fluorescein-12-dUTP incorporation during PCR amplification, and reverse probing, where the PCR product is used as the probe in a scaled down hybridization with a fixed capture probe consisting of a fragment entirely internal to the sequence of the PCR product. These techniques have shown sensitivities of 20 fg of purified mycobacterial DNA, which corresponds to approximately five cells.


Biopsy of canine ear tissue for detection of Borrelia burgdorferi following Ixodes dammini feeding.

A punch biopsy method was developed to detect and cultivate Borrelia burgdorferi from canine ear tissue. The biopsy procedure was sensitive in that spirochetes were detected in 100% of the dogs upon which infected ticks had fed. B. burgdorferi could be cultured as early as 6 days after tick feeding at which time the organisms could not be concurrently recovered from peripheral blood. The ear punch biopsy provides a reliable method for sampling live canines and should prove useful for detecting early B. burgdorferi infection and for monitoring duration of non-disseminated infection. Additionally, the procedure will aid in the development of an animal model for evaluation of preventive and therapeutic treatment modalities.


Effects of ethanol on the growth and elongation of Escherichia coli under high pressures up to 40 MPa.

The effects of ethanol on growth, viability and cell length were studied in Escherichia coli cultured under pressures up to 40 MPa (400 bar). A pressure of 10 MPa reversed the effect of ethanol in retarding cell growth. Cells cultured in the absence of ethanol became about seven times longer at 40 MPa than at atmospheric pressure, and some cells showed incomplete cell division. Ethanol also increased cell length but these effects were not seen at pressures of 20 MPa or more.


Management of allergies to animals.

Allergy to animals affects between 15 to 30% of atopic persons and about the same proportion of all those who work with laboratory animals. The responsible allergens are contained in the urine, saliva, and secretions of furred animals. The allergens apparently dry on fur, bedding, or other fomites so that with disturbance, the antigen becomes airborne on particles or "vectors" of diverse size. Many of these vectors are small enough to remain airborne and reach the lower airway. They adhere to surfaces in the environment such as rugs, walls, and clothing, so that animal allergens are found in virtually every sample of household dust and in the air and surfaces throughout most research facilities. This creates a reservoir of allergen that makes total elimination of the allergen from any environment very difficult. It then becomes important to understand the minimum level of allergen capable of inducing symptoms in sensitized patients so strategies can be developed to reduce exposures to below these limits. Otherwise, management will depend on nearly complete avoidance of animal dander, a difficult task. Alternatively, it will be necessary to modify the allergic host with drugs or immunotherapy.


Mast cell disease.

The mast cell, equipped with enzymes, chemotactic factors, a vasoactive amine, an anticoagulant, and lipid-derived proinflammatory products, may be essential in tissue modeling as well as in defense. Its primarily perivascular location in skin and the mucosa of the respiratory tract and the gut assures its availability to counter parasites. By the same token, the mast cell is responsible for interactions with inhaled, ingested, and injected antigens that comprise IgE-mediated allergic reactions. Abnormally high numbers of mast cells in the skin, either localized or generalized, result in urticaria pigmentosa or generalized cutaneous mastocytosis, respectively. Tissue infiltration by excessive mast cells, primarily in gut, bone, liver, and spleen, results in systemic mastocytosis; this may be accompanied by myelodysplasia or lymphoma and may eventuate in mast cell leukemia. Until the etiology of mastocytosis is understood, the treatment is symptomatic: histamine antagonism by H1 +/- H2 blockade for flushing, itching, and gastric distress; cyclooxygenase inhibition to prevent prostaglandin D2 (PGD2)-induced hypotension when indicated; and oral cromolyn to prevent gastrointestinal symptoms and bone pain.


Age and sex-ratio expression in Drosophila mediopunctata.

The sex-ratio trait--production of progenies with excess of females due to X-linked meiotic drive in parental males--has a variable expression in Drosophila mediopunctata. We tested the effect of male age and found that aging increases the expression of sex-ratio, a fact relevant for the interpretation of field data and for experimental design.


Localization of genes ecs, dor and swi in eight Drosophila species.

A cluster of genes corresponding to the early ecdysone stimulated puff 2B of the Drosophila melanogaster X chromosome has been localized using in situ hybridization in eight Drosophila species. Genes ecs, dor and swi from this cluster have been mapped in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum and D. paranaensis to the telomeric region of the X chromosome, in D. kanekoi to the distal region, and in D. pseudoobscura, to the proximal region of the X chromosome. It is assumed that organization of this cluster in these species is conserved. In D. hydei, multiple hybridization sites of certain DNA probes from this region were found.


Influenza vaccination: a successful outpatient program.

We initiated a program to evaluate patient reasons for refusing immunization in an allergy clinic. A general medicine clinic was studied for comparison. For the Northwestern University Allergy Service (NUAS) there were five full-time salaried and seven voluntary physicians. In the general medicine clinic there was one part-time salaried physician. Four hundred eighty-eight NUAS patients and 48 general medicine patients were evaluated. Ninety-five percent of the patients agreed to vaccination. Egg allergy, the only valid contraindication to influenza vaccination, was reported by three (< 1%) patients. Transient mild symptoms consisting of fatigue, myalgias, rhinitis and/or diarrhea were reported in 20% of the patients who received vaccination. No severe systemic reactions were reported. No significant difference in the vaccination acceptance rate was noted between the subspecialty and primary care outpatient clinics. The importance of influenza immunization in patients with asthma and in other high-risk populations deserves emphasis, and high success rates are achievable when emphasized by physicians.


Immunization update.

During the 1970s and the early 1980s, immunization practices in the United States were unchanged. Immunization against pertussis, tetanus, diphtheria, measles, mumps, rubella, and polio were routinely administered to children. Infections with these organisms declined dramatically. Nonetheless, research was vigorous, culminating in the 1980s in new vaccines and changes in immunization strategies and practices. This presentation will focus on these changes: universal hepatitis B immunization; two-dose schedule for the measles, mumps, rubella (MMR) vaccine, Hemophilus influenza type B vaccine for infants, acellular pertussis vaccine as booster immunizations, the inactivated polio vaccine, and the yet-to-be-licensed live varicella vaccine.


IgG subclass deficiency.

IgG subclasses have been recognized since the early 1960s. Four such subclasses, designated IgG1, IgG2, IgG3, and IgG4, are known to exist. Approximately 65 to 70% of the total circulating IgG in normal persons is of the IgG1 subclass. IgG2 constitutes 20 to 25% of circulating IgG, and IgG3 and IgG4 each represent less than 10%. Deficiencies in the various IgG subclasses have been detected in adults and children with common variable hypogammaglobulinemia as well as in those with relatively normal total IgG levels. An important issue facing clinicians today is to determine what, if any, therapeutic implications are associated with demonstration of an IgG subclass deficiency.


Human handedness and the concept of developmental stability.

A model is proposed to explain the etiology of pathological handedness. Developmental instability, caused by elevated genotypic homozygosity, environmental disturbances, or their interaction, overrides programmed laterality and handedness in the same way that it perturbs the bilaterally symmetrical expression of morphological and metric traits. The model predicts that pathological handedness should be elevated among individuals with higher than average homozygosity and individuals who have developed under unfavorable uterine environments. Suggestions are offered for specific populations in which the predictions may be tested.


Cytokines: clinical potentials for the allergic patient.

Classic allergic responses occur as a result of mast cell-bound IgE being cross linked by allergen, causing degranulation and activation with release of multiple biologically active mediators. Such mediators may have a direct effects on target tissues and/or promote the inflammatory milieu typical of late-phase responses. The production of IgE is a normal T-cell dependent antibody response. It is the specificity for allergens that is aberrant, resulting in a hypersensitivity state. Recent work has demonstrated that small molecular weight substances called cytokines are responsible for many immunological activities such as IgE production, mast cell and eosinophil maturation, and proinflammatory mediators that directly contribute to the pathology of late-phase allergic responses. Differences are being sought in the relative production of various cytokines that can be correlated with disease activity. This should find clinical use in diagnosis, prognosis, and/or monitoring of specific immunotherapy. Additionally, therapeutic agents are being sought that have various agonist/antagonist properties to correct aberrant cytokine production. Such agents likely will have a central role in future therapy for allergic diseases.


Characterization of latex antigen and demonstration of latex-specific antibodies by enzyme-linked immunosorbent assay in patients with latex hypersensitivity.

Two latex antigens, one extracted from surgical gloves (GE) and the other from the sap of Hevea brasiliensis plant (RPE) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. These antigens were used to detect latex specific IgE and IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in the sera of patients with meningomyelocele (spina bifida) and of normal controls. Of the 36 patients studied, 26 had wheal-and-flare skin-prick test reactivity to latex antigen with 11/26 having had a history of anaphylaxis to latex products. Twenty three of the 26 sera from skin-test positive patients and 10/11 patients with history of anaphylaxis demonstrated significant levels of latex specific IgE and IgG in the sera, whereas only 1/14 normals showed significant antibodies to latex. The remaining 10 patients, all skin-test negative with latex antigens, showed only low levels of antibodies. The findings indicate that the ELISA used in the present study employing partially characterized antigens has sensitivity and specificity to detect latex specific antibodies in the sera of suspected patients and can be used for presumptive diagnosis of latex allergy.


The SbcCD protein of Escherichia coli is related to two putative nucleases in the UvrA superfamily of nucleotide-binding proteins.

The derived amino-acid sequences of the proteins encoded by E. coli genes sbcC and sbcD have been compared with other protein sequences using computer assisted methods. This work has shown that SbcC and D, which inhibit the propagation of replicons containing long palindromic DNA sequences, are distantly related to two putative bacteriophage nucleases. These nucleases both comprise two polypeptide chains which are the products of genes 46 and 47 of bacteriophage T4 (gp 46 and gp 47) and genes D13 and D12 of bacteriophage T5 (gp D13 and gp D12). The comparisons reveal that SbcC, gp 46 and gp D13 are more closely related to each other than are SbcD, gp 47 and gp D12. SbcC appears to have undergone a partial duplication of an ancestral sequence. These proteins all contain motifs common to the superfamily of nucleotide-binding proteins that includes UvrA and the cystic fibrosis transmembrane regulator CFTR.


Fauna, flora, fowl, and fruit: effects of the Columbian Exchange on the allergic response of New and Old World inhabitants.

The Columbian Exchange has been described as "the most important event in human history since the end of the Ice Age." This interchange of many species of fauna, flora, fowl, and fruits resulted in new encounters between New and Old World inhabitants. Prominent among these were manifestations of allergic reactions to many of the new substances. Little imagination is required to reflect on what these substances, added to or detracted from both the New and Old World lifestyles, habits, and diets. The numerous peas, vegetable seeds, and grasses, such as sugarcane, introduced during Columbus' later voyages, made an enormous difference in the lives of New World inhabitants, as did the introduction of the cow and horse, not to mention substances such as coconuts and bananas, that are now intimately associated with the Caribbean and the Bahamas. This article focuses on some the more important exchange substances and emphasizes many forms of anaphylaxis: asthma, food allergy, hypersensitivity pneumonitis, chronic bronchitis, rhinitis, serum sickness, and other conditions that developed in both New and Old World inhabitants. To mention only a few examples, the Europeans introduced to the New World potential dangers such as honeybees (anaphylaxis). It also gave the New World the cow and the horse (serum sickness), which became the constant companion of Columbus' Indians and the American cowboy. It gave the Italians their thick red gravy, and the New World its pizza (food allergy). The Caribbean received bananas and coconuts and the New World embraced coffee (caffeine addiction). On the other hand, the exchange also caused Europeans to begin puffing away on tobacco.


Partial albinism, immunodeficiency, and progressive white matter disease: a new primary immunodeficiency.

We studied 12 children who presented with a recently recognized syndrome. The salient features of this new syndrome were recurrent fever; hepatosplenomegaly; pancytopenia; blond, golden to silvery gray hair; hypopigmented skin, progressive white matter demyelination; and early death. Seven patients died, four with severe central nervous system (CNS) involvement, and three with bone marrow failure and sepsis. Cutaneous anergy to recall antigens was present in all patients. Other immunological abnormalities were poor antibody responses; deficient T-cell responses to phytohemagglutinin (PHA), concanavalin A (Con-A), and allogeneic lymphocytes; hyperresponsiveness of B lymphocytes to pokeweed mitogen; and variable phagocytic defects. Histopathologic examination of the hair and skin biopsies showed characteristic distribution of melanin with melanocytes present in normal numbers but with fewer short dendritic processes. Langerhans' cells were present in normal numbers in some patients and sparse in others. This syndrome seems to cluster into two tribes from two different geographical areas in the Arabian Peninsula. In the eight families studied, 12 other siblings and close relatives were found to be affected. The mode of inheritance in this syndrome is that of an autosomal recessive pattern. We propose the term "PAID syndrome" to identify patients with the above features.


Peripheral nervous system in the larynx. An anatomical study of the motor, sensory and autonomic nerve fibers.

The laryngeal peripheral nervous system is presented on the basis of our results in the cat, following 10 years of investigation using mainly tracer techniques. The present paper focused on the localization of each laryngeal motoneuron, the myotopical arrangements of motoneurons innervating the pharyngeal and esophageal striated muscles within the nucleus ambiguus in the motor nerve supply, and also on the location of neurons, the distribution and density of nerve fibers, the area and laterality of the innervation, and the pathway to the larynx in the sensory and sympathetic nerve supplies. Regarding the parasympathetic nerve supply, the neural ganglia and the ganglionic cells in and around the laryngeal nerves and in the laryngeal framework are demonstrated. Most of this innervation, however, is still unclear. Discussions from the literature are also reported.


Examining the effects of amplification on the nasalance ratios of hearing-impaired children.

Nasalance scores were obtained for hearing-impaired children. Data speech samples obtained with and without personal amplification were compared for differences between gender and chronological age. Statistical comparisons are presented for each amplification condition. Significant differences for nasalance in the aided and unaided conditions were not evident although a high, positive correlation between conditions was reported. Nasalance, age and gender were not positively related. Dispersion of the nasalance scores for the two speech conditions was suggestive of persisting difficulties with velopharyngeal control during speech activities; mean scores revealed objective evidence of hypernasality.


[Electromyography of the larynx with skin surface electrodes].

A new method of skin surface electromyography (EMG) was evaluated. Twelve electrodes were placed symmetrically in the cricothyroid and thyroid lamina region. Raw EMG signals were continuously recorded, filtered, and rectified. Phonation of the vowel /a/ triggered a computer, which thereafter stored a 625-ms EMG recording, starting 375 ms before and ending 250 ms after onset of the audible phonation. Sixteen to 64 of these recordings were averaged to eliminate random noise. Twenty-six healthy subjects and 9 patients with unilateral laryngeal paralysis were studied. EMG activity exhibited great intra- and interindividual variation. Often, even in healthy subjects, EMG levels were different between the left and right side of the larynx. Thus, this method is not suitable for diagnosis or follow-up of laryngeal paralysis. Sources of variation in EMG activity are discussed.


Computerised measurement of fundamental frequency in Scottish neoglottal patients.

An electrolaryngograph has been used to investigate the fundamental frequency of neoglottal speech produced by patients who have undergone near-total (parsimonious) laryngectomy. The resulting estimates of fundamental frequency were compared with the fundamental frequency of normal and oesophageal speakers of a similar age and Scottish dialect. It was found that the average fundamental frequencies of neoglottal and oesophageal male speakers were higher than those in the normal group, though many fall into the normal range. The fundamental frequency of the single neoglottal female speaker was higher than the normal average, but the fundamental frequencies of all of the oesophageal female speakers were lower than those of the normal female speakers. The 80 and 90% ranges of the alaryngeal speakers were wider than those of the normal speakers, but the average range of neoglottals was narrower than that of the oesophageals for the male speakers.


Myofunctional therapy in patients with orofacial dysfunctions affecting speech.

Tongue thrusting, deviate swallowing, mouth breathing, orofacial muscle imbalance, deviate mandibular movement and malocclusion are the most important orofacial dysfunctions underlying disorders of articulation. Their development is linked to early bottle feeding and sucking habits. The phoniatrician is charged with the early detection of orofacial dysfunctions affecting speech. Early correction of habits and retraining by speech therapy are important preventive measures. Case histories, phoniatric and myofunctional diagnoses and dental/orthodontic findings were compiled for a total of 103 patients aged 3-30 years (11 +/- 4 years). Forty-five patients have completed a regimen of myofunctional therapy. For these patients highly significant improvements in lip strength, lip closure, breathing and tongue placement as well as in the swallowing pattern and orofacial muscle balance have been observed. Concomitantly, two thirds of the patients (66%) attained normal articulation. Speech defects were resistant to therapy in only 2 cases. In dental/orthodontic practice myofunctional therapy is used for retraining abnormal positions and functions of the orofacial muscles so as to create a normal occlusal relationship. The results of this study show that myofunctional therapy is highly instrumental also in phoniatrics as a special form of treatment for disorders of articulation.


Potassium channel openers: pharmacological and clinical aspects.

Opening of plasmalemmal K+ channels leads to cellular hyperpolarization which, in excitable tissues possessing voltage-dependent Ca2+ channels, prevents the opening of such channels and thus prevents excitation. In the last few years, an increasing number of compounds have been identified which elicit their effects by opening K+ channels, preferentially in smooth muscle, but also in other excitable tissues. These include the novel benzpyrans, cromakalim and bimakalim, the thioformamide aprikalim, and also well known antihypertensives such as minoxidil sulphate, diazoxide and pinacidil. After a short overview of the various families of K+ channel openers (KCOs), their basic pharmacological properties, including inhibition by the sulfonyl ureas (such as glibenclamide) are presented. The actual discussion concerning the type of K+ channel(s) opened by these compounds and their mechanism(s) of vasorelaxation will be reported. The therapeutic potential of these compounds in the cardiovascular field (as antihypertensives and, in particular, as anti-ischemic agents in heart and skeletal muscle), and in asthma (where they reverse established airway hyperreactivity) will also be discussed. Improved tissue selectivity may be the essential pre-requisite for true clinical success of this class of compounds.


KATP channels modulate GABA release in hippocampal slices in the absence of glucose.

We studied the effects of KATP channel blockers on [3H]GABA release in the absence of glucose in rat hippocampal slices. The omission of glucose induced a marked increase in the efflux of [3H]GABA, which was antagonized by TTX (1 microM), but not by MK 801 (1 microM) or DNQX (100 microM). Glibenclamide (10-100 microM) increased dose-dependently the release of [3H]GABA evoked in the absence of glucose. An increase in [3H]GABA release was also observed with gliquidone (100-300 microM), another sulfonylurea. The potentiation of [3H]GABA release induced by glibenclamide (100 microM) was antagonized by DNQX but not by MK 801. Thus, in the absence of glucose, KATP channel blockers enhance the release of GABA from rat hippocampal slices; this effect seems to be mediated by an overstimulation of non-NMDA glutamate receptors. On the basis of results reported in the present paper, we suggest that KATP channels may play a role in the regulation of GABAergic activity during hypoglycemia.


Effects of paraldehyde on the convulsions induced by administration of soman in rats.

The ability of paraldehyde, a potent central nervous system depressant, to prevent the convulsions induced by the organophosphate soman, an irreversible inhibitor of acetylcholinesterase, was studied in rats. Paraldehyde (0.1-500 mg/kg, im) administered 10 min before soman (100 micrograms/kg, sc) did not protect against seizures. Co-administered with atropine sulfate (10 mg/kg, im), paraldehyde produced a clear dose-dependent anticonvulsant response. Although this pre-treatment could delay the occurrence of death, it did not produce any change in the soman-induced 24 h mortality rate. Thus, co-administration of paraldehyde and atropine sulfate might constitute a valuable tool to be used against the convulsant consequences of soman poisoning. However, supplementary pre-medication, in addition to paraldehyde and atropine sulfate, remains necessary to improve the antilethal capacity of the pre-treatment.


A French version of the Sickness Impact Profile (SIP): stages in the cross cultural validation of a generic quality of life scale.

The Sickness Impact Profile is a quality of life scale developed in the United States which is now widely used in clinical trials in chronic conditions such as chronic obstructive lung disease, angina, rheumatoid arthritis, chronic pain, psoriasis, inflammatory bowel disease and cancer. Validated generic scales permit cross cultural comparisons within disease processes in clinical trials. As a simple, direct translation of a scale is inadequate, we have created a French version of the original US version of the Sickness Impact Profile. The first phase was qualitative. A first French translation of the original US version was back translated into English by three independent translators. A second French version resulted from a consensus reached by a panel of lay subjects and health professionals after comparing the original US version, the first French version and the three back translations. This second version was tested with a group of 40 healthy volunteers. This qualitative phase resulted in a French Test Version with content and face validity. The quantitative phase assessed the equivalence of the rank order of each item, by sub scale, in the US version with that of the French Test Version, ie the rank of the French item was comparable (+/- 2) to the rank of the corresponding US item. The French Test Version was tested with 47 healthy subjects. Of the 136 items of the Sickness Impact Profile, 13 were retranslated to create a final French version. This was similarly tested on ten healthy subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


Twenty-four-hour serum growth hormone, insulin, C-peptide and blood glucose profiles and serum insulin-like growth factor-I concentrations in women with polycystic ovaries.

Raised insulin levels are now recognized as a characteristic feature of women with polycystic ovaries (PCO), and hyperinsulinism has been shown to stimulate androgen production in such women. We have, however, recently shown that hyperinsulinaemia is present only in the obese subjects with PCO in whom insulin concentrations correlate with those of luteinizing hormone. We therefore studied 24-hour blood profiles of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in obese and non-obese women with PCO, for comparison with their levels of insulin, C-peptide and other hormones, such as androgens which are known to be disturbed in PCO. Mean 24-hour GH levels were higher overall in PCO than in control subjects, although the difference was not significant. When, however, a separate analysis was made in obese as compared with non-obese PCO patients, GH concentrations were significantly higher in the non-obese group than in the obese (p = 0.0005). There was a significant negative correlation between body mass index and mean 24-hour GH concentrations (r = -0.641; p = 0.0006). IGF-I concentrations were however similar in the PCO group overall and in controls, as well as in the obese and non-obese PCO patients. The 24-hour blood glucose profile pattern was significantly different in PCO women from controls (p = 0.009), with absence of post-prandial peaks in blood glucose concentrations. These changes were most marked in the non-obese PCO group, who also had significantly lower blood glucose levels than either controls or obese PCO subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


Menstrual variation in salivary testosterone among regularly cycling women.

To determine menstrual variation in salivary testosterone daily saliva samples were collected from 20 regularly cycling women. Results indicate that the menstrual profile of salivary testosterone for both ovulatory and anovulatory cycles exhibits local peaks during the follicular phase and at midcycle, as well as a luteal trough. However, the testosterone profile for anovulatory cycles exhibited a later midcycle peak than that for ovulatory cycles, as well as significantly higher average testosterone levels. These results extend the observation of a midcycle peak in serum testosterone to saliva and suggest the existence of a follicular peak in unbound testosterone coincident with the early androgen production of a cohort of developing follicles.


Effects of prolonged treatment with diltiazem on pituitary secretion of luteinizing hormone, follicle-stimulating hormone, thyrotropin and prolactin.

In previous studies it has been observed that acute administration or short-term treatment with calcium channel blockers can influence the secretion of some pituitary hormones. In this study, we have examined the effect of the long-term administration of diltiazem on luteinizing-hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH) and prolactin (PRL) levels under basal conditions and after gonadotropin-releasing hormone (GnRH)/thyrotropin-releasing-hormone (TRH) stimulation in 12 subjects affected by cardiovascular diseases who were treated with diltiazem (60 mg 3 times/day per os) for more than 6 months and in 12 healthy volunteers of the same age. The basal levels of the studied hormones were similar in the two groups. In both the treated patients and the control subjects, a statistically significant increase (p < 0.01) in LH, FSH, TSH and PRL levels was observed after GnRH/TRH administration. Comparing the respective areas under the LH, FSH, TSH and PRL response curves between the two groups did not present any statistically significant difference. These findings indicate that long-term therapy with diltiazem does not alter pituitary hormone secretion.


Knemometry in the assessment of short-term linear growth in a population of healthy school children.

Short-term lower leg length increments were monitored with weekly knemometry measurements during 3 months in 27 healthy school children aged 8-12 years. One year after the first visit the children were measured once again. The mean short term velocity of lower leg growth was 0.40 mm/week (SD 0.12 mm/week). The mean short-term and annual ratios between height velocity and lower leg growth velocity were identical (2.8). The relative variation between two observers was 0.08%. Knemometry is a suitable method for monitoring short-term linear growth in populations of children. Two trained observers may substitute for each other in group studies.


Final height after growth hormone therapy in peripubertal boys with a subnormal integrated concentration of growth hormone.

The aim of this study was to test the effect of growth hormone (GH) therapy on final height in peripubertal boys with idiopathic short stature in whom a subnormal integrated concentration of GH (< 3.2 micrograms/l) was found. Twenty-eight peripubertal children were studied. Height was below 2 SD for age, growth velocity was < 4.5 cm/year, bone age was more than 2 SD below mean for age and GH response to provocative tests was more than 10 micrograms/l. Eleven subjects (group B) were treated with recombinant GH 0.75 unit/kg/week, divided into 3 weekly doses for 2 years, and then the same weekly dose divided into daily injections was administered until final height was attained. Seventeen untreated children (group A) who were followed until cessation of growth served as controls. The GH-treated patients reached their target heights (-2.1 +/- 0.5, mean +/- SD in SDS) and predicted heights (-1.8 +/- 0.8) determined by the Bayley and Pinneau method, while the final heights of the untreated patients were significantly lower than their target heights and their predicted final heights (-2.7 +/- 0.7, -1.8 +/- 1.0 and -2.7 +/- 0.7, respectively). The main effect of GH was observed during the 1st year of treatment when height velocity was significantly higher in the GH-treated group than in the untreated one (9.3 +/- 2.1 vs. 5.3 +/- 1.1, respectively, p < 0.001). The high cost of the treatment in this specific age group should be weighed against the results.


Serum prolactin in patients with Laron-type dwarfism: effect of insulin-like growth factor I.

Prolactin (PRL) secretion was studied in Laron-type dwarfism (LTD) patients (8 children and 9 adults) in basal condition, after acute insulin-like growth factor (IGF-I) or TRH injections and during 2 months of daily IGF-I treatment. Basal PRL was repeatedly higher (12.6 +/- 1.6 micrograms/l) than that in control subjects (7.6 +/- 1.2 micrograms/l, p < 0.05). Acute IGF-I injection caused an immediate slight decrease in serum PRL and growth hormone (GH), followed by a progressive rise to mean peak levels of 33.3 +/- 4.5 micrograms/l again parallel to serum hGH which rose to 86 +/- 20 micrograms/l--a response to the IGF-I-induced hypoglycemia. Intravenous TRH in LTD children induced a marked response in serum PRL, similar to that registered in estrogenized adult females. Serum PRL did not show consistent changes during chronic IGF-I treatment. It is suggested that the higher-than-normal PRL levels and release in LTD patients are due to a drift phenomenon of the mammosomatotropes which produce large amounts of hGH.


Direct measurement of immunoreactive renin during changes of posture in man.

For several years, it has been possible to determine renin by a direct RIA. In the present study, plasma active renin concentration (PRC) was related to plasma renin activity (PRA) and aldosterone as a function of a standardized posture test. Using PRC, our target was to define the shortest necessary test duration. The three parameters were examined in 10 healthy male subjects (22-34 years old). Salt balance was determined in 24-hour urine, and plasma potassium and sodium were measured. Volunteers were hospitalized for 1 night, and at 8 a.m. the next morning they were subjected to the following postural changes: 3 h active orthostasis and 3 h recumbency. Frequent blood samples were taken. Orthostasis induced a significant rise in PRC, PRA and aldosterone already after 15 min. PRC and PRA reached a maximum level after 90 min of orthostasis and remained relatively stable, while aldosterone reached its highest level already after 30 min and then gradually decreased. Significant correlations were found between PRA and PRC (p < 0.001), between PRC and aldosterone (p < 0.001), and between PRA and aldosterone (p < 0.001). The PRC/PRA ratio changed during the course of the test, especially in supine subjects. When subjects returned to the supine position, all the parameters measured began a continual decrease. There were no significant changes in serum potassium and sodium levels throughout the duration of the test.(ABSTRACT TRUNCATED AT 250 WORDS)


HLA haplotypes and susceptibility to Graves' disease.

Graves' disease is a polygenic disease in which the HLA cluster could play a role. The purpose of our study is to identify HLA haplotypes in a family with closely related susceptibility to Graves' disease and foresee the risk of disease in the youngest daughter. The family studied had included the father (47 years), mother (46 years) and 3 daughters (18, 17 and 13 years). The mother and 2 eldest daughters were affected by Graves' disease. HLA-A, -B, -C, -DR and -DQ were performed with standard microlymphotoxicity techniques. A mother's role in passing susceptibility to Graves' disease to daughters is undisputed; it seems to be due to the B35 HLA allele. Also, the third daughter (at 15 years) has an HLA B35 allele, and actually has an incipient humoral hyperthyroidism.


Relation between nocturnal melatonin profile and hormonal markers of puberty in humans.

We examined the relation between nocturnal melatonin and hormonal markers of puberty in 57 normal children and adolescents and 39 subjects with disorders of pubertal onset. Melatonin was measured in hourly blood samples drawn overnight by constant withdrawal. Basal 08.00 h plasma testosterone, estradiol and LH, and the peak LH response to LHRH administration were determined. There were no significant correlations between testosterone, estradiol, basal LH and peak LH and melatonin peak (r = -0.18, -0.22, -0.02, -0.12, respectively) or melatonin peak time (r = 0.12, -0.01, -0.02, 0.07 respectively). The results were not affected significantly by sex, diagnosis or age. A comparison of subjects grouped by peak LH < 15 U/l (most likely prepubertal; n = 40) and peak LH > 30 U/l (most likely pubertal; n = 34) showed no significant differences in melatonin peak (160.5 +/- 59.3 vs. 146.6 +/- 50.9 pg/ml; t = 1.09; p > 0.05) or melatonin peak time (1.8 +/- 1.7 vs. 2.5 +/- 1.7 h; t = -1.79; p > 0.05). Although a pineal-puberty relation cannot be excluded, the results do not support the hypothesis that melatonin restrains the hypothalamic-pituitary-gonadal axis during childhood.


Normal hemodynamic and coagulation responses to 1-deamino-8-D-arginine vasopressin in a case of lithium-induced nephrogenic diabetes insipidus. Results of treatment by a prostaglandin synthesis inhibitor (indomethacin).

The effect of 1-deamino-8-D-arginine vasopressin (DDAVP) on mean arterial pressure, pulse rate (PR), plasma renin activity (PRA), plasma factor VIIIc and von Willebrand factor were studied in a case of persistent lithium-induced nephrogenic diabetes insipidus (LINDI). 20% decrease in MAP, 22% increase in PR, 100% in PRA, and release of coagulation factors (2- to 3-fold) were noticed after infusion of 0.3 micrograms/kg DDAVP. Urinary prostaglandin (PG) E2 were enhanced. The treatment of this LINDI by PG synthesis inhibitor (PSI) combined with a low osmotic diet (LOD) led to a 51% fall in urine volume, 57% in free water clearance and 75% in sodium clearance. Urinary osmolality rose by 42% but remained low, probably in part because of the LOD. Urinary PGE2 was about one fifth of the initial high value. The results argue for (1) an end-organ resistance to DDAVP confined to the kidneys in LINDI and (2) an effectiveness of indomethacin combined with an LOD.


Stimulation of nitrogen and whole-body protein metabolism in growth hormone-deficient children by recombinant human growth hormone: relationship to growth.

The effect of a mammalian-cell-derived recombinant human growth hormone (rhGH) on nitrogen and whole-body protein metabolism was assessed in 12 children with complete growth hormone (GH) deficiency. All the patients received single oral doses of 15N-glycine (95 atom % 15N), 20 mg/kg body weight, prior to and following 7 days of treatment with rhGH, 1.7 IU/m2 body surface area (BSA) per day, administered subcutaneously. Prior to rhGH, mean urinary 15N-nitrogen excretion was 42.8 +/- 8% of the administered dose, which fell significantly to 22.8 +/- 7% during rhGH administration (p < 0.0001). Stimulation of protein metabolism by rhGH resulted in a protein net gain rate of 1.1 +/- 0.4 g/kg/day, which was significantly higher than the 0.6 +/- 0.5 g/kg/day rate seen prior to rhGH (p < 0.001). In patients subsequently placed on daily subcutaneous injections of rhGH 1.7 IU/m2 BSA, mean height velocity standard deviation score (HV SDS) for chronological age significantly increased from -3.8 +/- 2.6 to +8.5 +/- 3.1 and +3.3 +/- 2.2, during the 1st and 2nd years of treatment, respectively. However, there was no correlation between the long-term response to rhGH treatment and the short-term changes in nitrogen or protein metabolism in GH-deficient children.


Spanish multicenter clinical trial of recombinant growth hormone produced in mammalian cells for treatment of growth failure due to idiopathic growth hormone deficiency. Spanish Multicenter Study Group.

A total of 17 patients (14 boys, 3 girls) with poor growth due to idiopathic growth hormone deficiency (IGHD) were treated for 12 months with recombinant human growth hormone (rhGH; Saizen), 0.5 IU/kg body weight per week administered by subcutaneous injection on a daily basis. Therapy significantly increased growth velocity (GV) from 4.0 +/- 1.5 to 10.1 +/- 2.4 cm/year (p < 0.001). Correspondingly, GV standard deviation score (GV SDS) for chronological age (CA) increased from -2.7 +/- 1.6 to +4.5 +/- 2.8; GV SDS for bone age (BA) increased from -4.0 +/- 2.0 to +3.6 +/- 3.3; and patient height SDS-CA increased from -3.5 +/- 1.1 to -2.4 +/- 0.7. Changes in these parameters were significant at p < 0.001. During the course of the study, mean plasma IGF-1 levels rose significantly from 0.3 +/- 0.2 IU/ml to 1.3 +/- 1.2 IU/ml (p < 0.01). BA maturation proceeded at approximately 1 year per treatment year. Therapy was well tolerated. Anti-hGH antibodies were seen in only 1 patient at a low concentration, and did not appear to affect GV. This study demonstrates that rhGH is safe and efficacious when used to increase height in patients with IGHD.


Growth response to recombinant human growth hormone of mammalian cell origin in prepubertal growth hormone-deficient children during the first two years of treatment.

In five clinical studies performed in Austria, France, the FRG, Italy, Switzerland, the UK and the USA, 304 growth hormone (GH)-deficient children were treated with recombinant human GH (rhGH) of mammalian cell origin. Two hundred and twenty-five patients were previously untreated (naive patients), and 79 were transferred from pituitary hGH after interruption of therapy for at least 6 months (transfer patients). Two treatment protocols, differing in both dose and frequency of injections, were used: (1) a dose of 0.6 IU/kg body weight per week was administered in 3 s.c. injections to 203 patients (178 naive, 25 transfer; group 1); and (2) a dose of 0.45 IU/kg body weight per week was administered in 7 s.c. injections to 101 patients (47 naive, 54 transfer; group 2). After 1 and 2 years of treatment, 143 and 109 naive, and 51 and 46 transfer patients, respectively, were still prepubertal, and their data were analyzed for efficacy. During the 1st year of treatment, both naive and transfer patients on daily injections (group 2) demonstrated better growth than those on 3 injections per week (group 1), with height velocities (HVs) of 10.6 +/- 2.7 cm/year (group 2) versus 8.6 +/- 2.0 cm/year (group 1) for naive patients (p < 0.001), and 9.9 +/- 1.9 cm/year (group 2) versus 7.2 +/- 2.7 cm/year (group 1) for transfer patients (p < 0.001). The corresponding changes in height standard deviation score (delta H SDS) for chronological age (CA) were +1.3 +/- 0.6 (group 2) versus +0.8 +/- 0.5 (group 1) for naive patients (p < 0.01), and +1.1 +/- 0.3 (group 2) versus +0.6 +/- 0.4 (group 1) for transfer patients (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Recombinant human growth hormone and oxandrolone in treatment of short stature in girls with Turner syndrome.

91 girls with Turner syndrome (TS) with a mean chronological age (CA) and bone age (BA) of 10.3 +/- 2.3 and 8.9 +/- 1.9 years, respectively, were randomly assigned to subcutaneous treatment with recombinant human growth hormone (rhGH) alone (n = 47), 2.6 IU/m2 body surface area daily or combination treatment (n = 44) with the same dose of rhGH and oxandrolone 0.1 mg/kg body weight orally, for the first 12 months of this study. During the 1st year of therapy, there was a striking increase in height velocity (HV) in both groups, from 4.0 +/- 0.8 to 6.3 +/- 1.3 cm/year [HV standard (standards of untreated Turner patients) deviation score (SDS) for CA from 0.0 +/- 0.7 to 2.9 +/- 1.3] in the rhGH group and from 4.2 +/- 1.2 to 8.5 +2- 1.7 cm/year (HV SDS-CA from +0.3 +/- 1.0 to 5.6 +/- 1.6) in the combination group. The difference between the groups was statistically significant (p < 0.01). During the 2nd year of treatment, the rhGH dose was increased to 3.4 IU/m2 daily for the rhGH-alone group, whereas in the combination treatment group the oxandrolone dose was reduced to 0.05 mg/kg daily. HV was maintained at significantly higher levels than those prior to treatment, at 5.3 +/- 1.1 cm/year (HV SDS-CA: +2.1 +/- 1.3) and 6.2 +/- 1.5 cm/year (HV SDS-CA: +3.6 +/- 1.4) in the rhGH-alone and the combination group, respectively (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Immunogenicity of a mammalian cell-derived recombinant human growth hormone preparation during long-term treatment.

The development of anti-human growth hormone (anti-hGH) and anti-host-cell protein antibodies to recombinant hGH (rhGH) of mammalian cell origin was determined in 395 children (304 GH deficiency; 91 Turner syndrome) undergoing long-term treatment (up to 54 months) for growth disorders. In all patients, blood samples were obtained prior to and every 2-3 months during treatment, and analyzed at a central laboratory for anti-hGH antibodies by RIA and antibodies to host-cell antigens by ELISA. During the first 24 months of treatment, 9 (3%) of the 304 patients with GH deficiency developed antibodies to rhGH for longer than 3 months. However, persistent antibodies were seen in only 2 patients, both of whom had proven hGH-N gene defects. In the remaining 7 (2%) anti-rhGH-antibody-positive patients, antibody concentrations showed a tendency to increase for 3-12 months, irrespective of the time of onset of measurable concentrations, and declined thereafter. In these patients, binding capacities were between 0.01 and 0.1 mg/l, and binding affinities were between 7 x 10(8) and 8 x 10(9) l/mol. Height velocity was unaffected in these children. None of the 91 patients with Turner syndrome developed persistent anti-hGH antibodies. Further, no child developed antibodies to host-cell antigens during treatment with rhGH of mammalian cell origin.


Pharmacokinetics and short-term metabolic effects of mammalian cell-derived biosynthetic human growth hormone in man.

The pharmacokinetics and acute effects of an authentic recombinant DNA-derived human growth hormone (rhGH) produced by genetically engineered mammalian cells were determined in 12 healthy volunteers following intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) administration of 4 IU (1.3 mg) hGH/m2 body surface area. Following i.v. administration, apparent elimination half-life of rhGH was 18 min. Following i.m. administration, a mean peak serum concentration of 36.9 ng/ml (range 13-61 ng/ml) occurred at 3 h, and following s.c. administration, more sustained but lower serum concentrations occurred, with mean peak concentrations of 16.4 and 16.3 ng/ml at 4 and 6 h (ranges 9.0-27.5 ng/ml and 6.5-35.5 ng/ml at 4 and 6 h, respectively). The mean area under the curves was lower after s.c. (134 +/- 48 ng.h.ml-1) than after i.m. (194 +/- 48 ng.h.ml-1) injections (p < 0.03). Comparable results were obtained for the same dose of rhGH given subcutaneously in concentrations of either 4 IU/ml or 10 IU/ml. Both i.m. and s.c. administrations caused similar increases in free fatty acids at 4 h and insulin-like growth factor I at 24 h. Insulin, C-peptide and blood glucose were almost unchanged during the first 4 h after administration, whereas leukocytes increased significantly (p < 0.0001). Local and systemic tolerance were good, and no adverse reactions were observed. In a GH-deficient child, hGH serum levels between 10 and 20 ng/ml were demonstrated for a period of 8 h after s.c. administration of 0.07 IU rhGH/kg body weight.


Improvement of metabolic control and changes of fatty acid composition do not alter platelet aggregability and thromboxane production in type 2 diabetes mellitus.

To evaluate whether improvement of metabolic control or changes in fatty acid composition of serum lipids may alter thromboxane (TXB2) formation and platelet function we followed up 25 newly diagnosed type 2 diabetics without angiopathy for about 6 months. Improvement of metabolic control was associated with significant decrease in total cholesterol (C), HDL-C, LDL-C, triglycerides (TG) and ratios of total C/HDL-C and LDL-C/HDL-C, respectively. Palmitic acid of TG and phospholipids decreased significantly whereas linoleic acid increased in the two serum lipids. The ADP-induced platelet aggregability and sensitivity were not altered. There was even no effect on TXB2 synthesis capacity of clotting whole blood during 6 months of treatment. Platelet aggregability and TXB2 formation were not correlated to the degree of metabolic control, nor was there any correlation to serum lipids and their fatty acid composition.


High urinary excretion of adrenaline in insulin dependent diabetic subjects.

In view of the hyperglycemic and ketogenic actions of catecholamines, studies on the adrenergic pattern in diabetic patients are relevant to the management of diabetes. We have studied urinary adrenaline and noradrenaline excretion in 4-hour collections in 16 type I diabetic male patients without signs of autonomic or peripheral neuropathy and 11 age-weight matched controls. In diabetic patients adrenaline levels were higher than control subjects (26.8 +/- 3.9 vs 10.7 +/- 3.0 nmol/4h; p < 0.003) but did not differ in respect of noradrenaline (62.6 +/- 6.8 vs 59.6 +/- 10.8 nmol/4h) and creatinine excretion. This finding demonstrates a hyperactivity of the adrenal medulla in diabetic patients without concomitant elevations of noradrenaline. This occurred in absence of hypoglycemia and seems to be a common feature of type I diabetics without complications. Since these levels of adrenaline are sufficient to stimulate both lypolysis and glycogenolysis a previous characterization of adrenomedullary activity may help to better define insulin demands in diabetic patients.


Responses of peripheral blood cells to acute insulin-induced hypoglycaemia in humans: effect of alpha-adrenergic blockade.

Acute hypoglycaemia provokes rapid changes in peripheral blood cell counts. To examine possible adrenergic mechanisms modulating these changes, counts of peripheral blood cells including lymphocytes, granulocytes and red cells were measured in response to acute hypoglycaemia in a group of six normal subjects in control conditions (study 1), and during alpha-adrenergic blockade with phentolamine (study 2). In study 1 hypoglycaemia provoked a biphasic white cell response, with an early rise in lymphocyte count and a later rise in granulocyte count. The red cell count increased modestly following hypoglycaemia. During alpha-adrenergic blockade, the rise in total white cell count was diminished, with the rise in the lymphocyte count being greatly obtunded. The rise in the granulocyte count was unchanged. The increment of the red cell count during acute hypoglycaemia was abolished. These findings suggest that the increments of peripheral lymphocyte and red cell counts in response to acute insulin-induced hypoglycaemia are mediated via alpha-adrenoreceptors.


Etiology and pathogenesis of type 1 diabetes.

An increasing bulk of evidence suggests that type 1 (insulin-dependent) diabetes mellitus is an autoimmune disease with a strong immunogenetic background. 1st-degree relatives of type 1 diabetic patients, especially HLA-identical individuals, bear an increased risk to develop the disease. The autoimmune reactions are pronounced at the onset of disease where an infiltration of islets with T and B lymphocytes, plasma cells and macrophages can be observed. Autoreactive T lymphocytes play a crucial role among effector mechanisms which finally lead to a selective destruction of pancreatic beta cells. Disease-specific autoantibodies (Ab) include cytoplasmic islet cell Ab (ICA), islet cell surface Ab (ICSA), Ab to the 64KD islet cell protein and Ab to insulin (IAA). As ICA can be detected months or years before the onset of clinical disease, testing of individuals at risk or population screening programs can help to recognize subclinical insulitis. High titers of ICA and high levels of IAA, as measured by radioimmunoassay, indicate a high risk for progression to type 1 diabetes. A blunted first phase insulin response in the i.v. glucose tolerance test is the most sensitive sign of an irreversible metabolic deterioration. It is likely that immunotherapy at a prediabetic state will be more efficacious than its initiation after the clinical manifestation of diabetes. However, the appropriate immunotherapeutical strategies are yet to be worked out.


A new ELISA measuring antibodies to islet cell membranes: levels are increased in type 1 diabetes.

Using purified rat islet cell membranes as the solid phase antigen we have established an ELISA to detect anti-islet cell membrane antibodies. Levels were increased in Type 1 diabetics compared to controls (p < 0.02). There was no relationship between the level of antibodies to the membrane and the presence of islet cell cytoplasmic antibody (ICA), suggesting that they are not the same. The assay offers the opportunity to study antibodies to human beta cell membranes, the target of the immune attack in type 1 diabetes.


A new ELISA assay for islet cell surface antibodies determination in type 1 diabetes mellitus of recent onset.

Sera from 46 newly diagnosed type 1 diabetic patients and from 100 normal controls were screened for ICA Cf ICA (standard immunofluorescence test) and for ICSA (ELISA assay) detection. Among 46 patients, 33 (71.7%) were ICA positive (16 of them also Cf ICA positive) and 21 resulted ICSA positive (45%). No correlation was found between ICSA, ICA and Cf ICA respectively. Therefore the ICSA detection, by a new and sensitive ELISA assay, in addition to ICA immunofluorescence determination, may be of significant diagnostic value in IDDM of recent onset.


CD11B lymphocytes in type 1 diabetes mellitus of recent onset.

To evaluate the behaviour of CD8+ cells (suppressor/cytotoxic T lymphocytes) and CD8+Leu15+ cells (CD11B, suppressor T lymphocytes) in type 1 diabetes mellitus, 27 newly diagnosed diabetic patients and 47 normal controls were studied. CD8 cells were significantly increased in 6 patients, decreased in 9 and unchanged in 12. In all diabetic patients was present a lack of CD8+ Leu15+ cells. These results show that in newly diagnosed type 1 diabetes mellitus, suppressor T lymphocytes are reduced independently from the number of CD8 cells.


C4 serum levels in newborns of diabetic mothers: relationship with metabolic assessment during pregnancy.

Serum levels of C4 complement factor are lower in diabetic patients: it is supposed that this finding is related to genetic inheritance factors of diabetes. The purpose is to compare C4 levels in newborns of diabetic mothers with maternal metabolic control during pregnancy. The results suggest that metabolic control could influence the expression of genetic factors of diabetes; in fact C4 serum levels in newborns are significantly higher (p < 0.01) than controls, but not different from those observed in large newborns of non diabetic mothers.


Insulin analogues with improved absorption characteristics.

The insulin preparations available today are not ideal for therapy as s.c. injection does not provide a physiological insulin profile. With the aim to improve the absorption properties recombinant DNA technology has been utilized to design novel insulin molecules with changed physico-chemical characteristics and hence altered subcutaneous absorption kinetics. Soluble, long-acting human insulin analogues in which the isoelectric point has been increased from 5.4 to approx. 7 are absorbed very slowly, providing a more constant basal insulin delivery with lower day-to-day variation than present protracted preparations. In addition they have better storage stability. Rapid-acting human insulin analogues with largely reduced self-association are absorbed substantially faster from subcutaneous tissue than current regular insulin and thus are better suited for bolus injection. The absorption kinetics of these analogues have been able to explain the mechanism behind the dose effect on insulin absorption rate.


Entero-insular axis and diabetes mellitus.

1. The incretin effect (i.e. the difference between the insulin response after oral and i.v. glucose) is reduced in type 2 diabetes although GIP secretion is normal or exaggerated. This suggests an insensitivity of the diabetic B-cell to GIP. However, it could also indicate the lack of another not yet defined "incretin". 2. While CCK is a potent incretin in rats and dogs, physiological concentrations of this hormone do not stimulate insulin secretion in man in presence of elevated blood levels of glucose or phenylalanine in the physiological range. It also does not interact with GIP. 3. Glucagon-like peptide I (7-36) is a potent glucose-dependent stimulator of insulin secretion in animals and man. Preliminary data suggest release after oral glucose despite localization of the GLPI containing cells predominantly in the ileum and colon. More data are needed before GLPI (7-36) can be regarded as a physiological incretin and its role in type 2 diabetes assessed.


Proinsulin as a possible therapeutic agent.

Gene technology has made significant amounts of biosynthetic human proinsulin (BHP) for biological and clinical studies available. It has been shown both in vitro and in vivo that BHP is about 10% as active as biosynthetic human insulin. More specifically it is 8% as potent regarding its action in stimulating peripheral glucose disposal and 12% as potent in suppressing hepatic glucose output. Its hypoglycemic effect is rather produced by the intact molecule or its intermediates and not by conversion to insulin or C-peptide. BHP appears to exert a more prolonged action when administered subcutaneously compared to NPH insulin. Furthermore because of its weaker effect on peripheral glucose utilization it is expected to create less profound hypoglycemia. Under conditions of equipotency BHP may stimulate triglyceride synthesis in a lower degree and in that way be less lipogenic. On longterm basis BHP proved to have a low immunogenic potential. We investigated the efficacy of BHP in split doses in type II diabetics with relative insulin resistance. Glucose levels at 1600 and 2000 and 0400 hrs as well as integrated glycemia over the 24 h period were lower under BHP. The potency of BHP in this and other studies has been demonstrated after application of greater quantities from it in terms of equimolarity. Nevertheless there are certain features such as the protracted action, the stronger effect on hepatic glucose production and the lower suppression of endogenous insulin secretion which make proinsulin a promising agent in the future management of type II diabetes.


Timing between the subcutaneous administration of insulin and consumption of a carbohydrate rich meal.

There is considerable uncertainty as to the appropriate timing of preprandial injections of short acting insulin before consumption of a meal. 8 healthy male volunteers were subjected to a glucose clamp (glucose 4.2 mmol/l) by means of a continuous insulin infusion on two occasions. On each occasion the subjects ingested a standardized meal. Twelve units of short acting insulin were injected subcutaneously either immediately before (A) or 30 min prior to the meal (B). In a control study (C) only the meal was consumed. The maximal increment of insulin concentrations after sc injection was 53.1 (6.9) mU/l in A and 58.6 (5.1) mU/l in B and thus comparable to C (52.8 (4.7) mU/l), however it was reached 30 to 60 min later as compared to endogenous stimulation. Injection 30 min prior to meal resulted in higher insulin concentrations early after the meal, but the glucose infusion rate doubled to 5.3 (0.3) mg/kg/min before the meal in order to prevent hypoglycaemia. Omission of this time interval is less hazardous, but bears the risk of late postprandial hyperinsulinaemia.


Experiences with a computerized diabetes information system.

This study shows the results of working with a computerized system which stores values of blood glucose, insulin, diet and exercise and demonstrates which diabetic patients are suitable for continuous monitoring. The recording of blood glucose as well as the insulin doses, diet and exercise correlated to time is more accurate and easier than the usual protocolling of data. Deficiencies in diet, exercise or insulin regimens are detected more easily by evaluation of data with a computerized system, for example by making an average profile of a day. These results have a strengthening effect: Well controlled diabetic patients are ambitious to further improve their blood glucose control. Poorly controlled diabetics appear to be frustrated by this kind of unfailing control.


Insulin delivery by implantable pumps.

We have developed a neutral human insulin (Hoe 21 GH) which is stabilized for use in implantable roller pumps. After extensive in vitro tests and animals experiments this preparation was selected for a clinical trial in humans. Twenty remote-controlled insulin pumps (Siemens AG) were implanted into insulin-dependent Type I diabetic patients for a one-year feasibility trial in four centres. The total observation time was 18.2 patient years. Three pumps had to be prematurely explanted after 101, 141 and 236 days. Blood glucose was measured by self-monitoring with 5.5 (1-17) values per day. 62.9% of these were in the range 3.33-8.88 mM. 3.25 glucose values per patient month were in the hypoglycemic range (lower than 2.78 mM) and 2.6 symptomatic hypoglycemias were reported per patient month, of which 0.22 per patient year required medical attention. The median HbA1c level was 7.6% at base line (5.9%-9.1%, 10-90% percentile) and 7.0% at the end of the trial (5.7%-8.3%, 10-90% percentile) (p < 0.05). Although some technical and clinical problems were observed, the study showed that the combination of this dosing device and a stabilized insulin preparation was a successful approach to the treatment of insulin-dependent patients.