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The respective influence of age, sex and health states on peripheral blood mononuclear subpopulations has been investigated in 194 institutionalized subjects. The "ill group' includes patients with various diseases and the "reference group' was referred to admission criteria for immuno-gerontological disease. A decline of total mature T-cell (OKT3) and helper T-cell (OKT4) proportions with ageing has been found only in the ill group and remains stable in the reference group. This age-dependent decline should represent either a susceptibility for illness or a consequence of higher incidence of illness with ageing. Suppressor-cytotoxic (OKT8), B- and T-activated (OKIa1), null (OKM1), and early E-rosette forming cells do not vary with ageing in both groups. It has been established previously that women have higher values of the percentages of early E-rosette forming cells.

The properties of follitropin receptors in immature porcine testis were determined using highly purified porcine follitropin. 1. The characteristics of follitropin binding to a subcellular fraction rich in plasma membranes were studied using a 125I-labelled follitropin with high specific activity (75-100 Ci/g) and high binding activity. The binding is dependent on time, temperature and pH. It is specific to follitropin as demonstrated by the very low binding activity of the follitropin alpha and beta subunits and of the other glycoprotein hormones. Scatchard analysis of binding data indicated an equilibrium association constant of 2 x 10(10) M-1 and a concentration of high affinity binding sites of 500 fmol/mg membrane proteins. 2. A sensitive radio-ligand receptor assay was developed. Fifty percent inhibition of binding was obtained with as little as 2 ng of porcine follitropin. Ovine and bovine follitropins and pregnant mare serum gonadotropin gave binding inhibition curves parallel to that given by porcine follitropin. With equine and human follitropin, significantly different slopes were recorded. 3. Kinetics of dissociation of labelled follitropin from its testis receptors showed the presence of at least two compartments with fast and slow dissociation rate constants. The ratio between the sizes of the slow and fast compartments appeared dependent upon preincubation time. 4. A temporal correlation was observed between binding of follitropin to testis receptors and activation of membrane bound adenylate cyclase.

The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action.

Hormone production was studied in situ by immunocytochemical methods in 20 pituitary adenomas. Special attention was given to 13 adenomas removed from acromegalic patients. Out of them, 6 had mild to moderate hyperprolactinemia. Immunohistochemistry revealed PRL-containing cells without any close relationship with high PRL serum level in 6 patients. Double immunogold staining revealed mammosomatotrope cells characterized by simultaneous presence of GH and PRL in the same granules in 2 patients. Since mammosomatotropes have never been demonstrated in the normal pituitary, our results probably signify gene dysregulation in pituitary cells of those acromegalic patients.

A new solid-phase enzyme immunoassay measuring the gelatin-binding capacity of plasma fibronectin has been developed. This assay is based on the direct and high-affinity interaction between fibronectin and gelatin coated to polyvinyl chloride plates. The amount of fibronectin bound to gelatin is then measured by sequential incubation with a specific rabbit anti-human fibronectin antiserum, with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies and with substrate. The final degradation of the substrate is read at 492-650 nm in an ELISA processor. The assay allows the accurate detection of fibronectin concentrations ranging from 1 to 20 micrograms/ml, is inhibited by the addition of gelatin to plasma, is highly reproducible (interplate CV less than 10%), requires 100 microliter of plasma only and has been fully automated. Significant linear correlations were noted between total antigenic fibronectin (measured by laser nephelometry) and fibronectin gelatin-binding capacity in plasma from 310 blood donors. Both parameters were higher in men than in women and significantly increased according to age. Dissociation between immunoreactive fibronectin and fibronectin gelatin-binding capacity was observed in two polytraumatized patients. This enzyme immunoassay therefore provides a new method to investigate functional alterations of the gelatin-binding domain of fibronectin in various pathological conditions.

Hypoxic stimulation of the peripheral chemoreceptors has been reported to inhibit hypoxic pulmonary vasoconstriction. To evaluate the pathophysiological importance of this observation, we investigated the effects of surgical peripheral chemoreceptor denervation on pulmonary vascular tone and gas exchange in 17 pentobarbital-anesthetized dogs with oleic acid pulmonary edema. Pulmonary arterial pressure-cardiac index (Ppa/Q) plots, blood gases, and intrapulmonary shunt measured by the SF6 method were obtained at base line, after peripheral chemodenervation (n = 9) or after sham operation (n = 8), and again after 0.09 ml.kg-1 intravenous oleic acid. Over the range of Q studied (2-5 l.min-1.m-2), Ppa/Q plots were best fitted as first-order polynomials in most dogs in all experimental conditions. Chemoreceptor denervation increased Ppa at the lowest Q, while sham operation did not affect the Ppa/Q plots. Oleic acid increased Ppa over the entire range of Q and increased intrapulmonary shunt. This latter was measured at identical Q during the construction of the Ppa/Q plots. Chemoreceptor-denervated dogs, compared with sham-operated dogs, had the same pulmonary hypertension but lower intrapulmonary shunt (36 +/- 4 vs. 48 +/- 5%, means +/- SE, P less than 0.04) and venous admixture (43 +/- 4 vs. 54 +/- 3%, P less than 0.02). We conclude that in intact dogs chemoreceptor denervation attenuates the rise in intrapulmonary shunt after oleic acid lung injury. Whether this improvement in gas exchange is related to an enhanced hypoxic pulmonary vasoconstriction is uncertain.

The amino acid sequences of both the alpha and beta subunits of porcine thyrotropin have been studied. Bovine thyrotropin primary structure was taken as a model for ordering the tryptic peptides of porcine thyrotropin. The amino acid sequence of the alpha subunit is identical to that of porcine luteinizing hormone, while oligosaccharide side-chains differ in composition. The primary structure of the beta subunit differs from that of bovine thyrotropin by six amino acid replacements, in positions 22, 24, 26, 36, 62 and 69, and by the absence of a methionyl residue at the carboxy terminus. Chemical evolutions of thyrotropin and luteinizing hormone are compared.

We have studied the specific effects of highly purified pituitary hormones on testicular cell multiplication and differentiation in the immature hypophysectomized rat. LH exhibits multiplicative effects on Leydig cells and, curiously, regressive effects on germ cells. Seminiferous tubules are the main site of action of FSH which induces direct differentiation of Sertoli cells and paracrine effects on germ cells resulting in their multiplication to the pachytene stage (in the absence of androgen). Through interstitium-directed paracrine effects, FSH promotes the differentiation of Leydig cells. Prolactin and lactogenic growth hormones are capable of exerting both differentiative and multiplicative effects on Leydig cells. The purity of the hormone preparations permit to define their specific and direct effects and those mediated by paracrine or autocrine factors synthetized locally under their control. Among these potential factors, we have demonstrated the pituitary-dependent expression of IGF-I, c-myc, c-fos proteins. The purity of the preparations used in this work permits to distinguish clearly the specific and direct effect of each pituitary hormone from those mediated by paracrine or autocrine factors.

Antisera were raised against highly purified preparations of porcine luteinizing hormone (pLH) and follicle-stimulating hormone (pFSH). Highly specific and sensitive radioimmunoassay systems were developed. The antisera to LH and FSH were used at working dilutions of 1:500,000 and 1:200,000 respectively and the sensitivities of the assays were 0.1 ng LH/ml serum (3 x 10(-12) mol/l) and 0.5 ng FSH/ml serum (1.5 x 10(-11) mol/l). The LH and FSH preparations used as standards were 1.2 and 81 times as potent as NIH-LH-S15 and NIH-FSH-P1 respectively. Both assays were validated and adapted for the measurement of the gonadotrophin content of porcine serum. The concentrations of LH and FSH in blood were measured simultaneously in prepubertal sows throughout a 24 h period, in adult sows during the oestrous cycle and in both prepubertal and adult animals after treatment with LH releasing hormone.

Highly specific radioimmunoassay systems were developed for measurement of hLH and hCG using antisera purified by affinity chromarography or simple adsorption to select the antibodies reacting specifically with either gonadotropin. Such systems permit specific measurement of lLH and hCG in samples containing both. These assays are suitable for various clinical and physiological studies, particularly, study of pituitary functions in the presence of chorionic or trophoblastic secretion.

Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.

The Fc-receptor (Fc-R) function of monocytes isolated from 19 control subjects and from 30 patients presenting with a rheumatoid arthritis (RA) was assessed in vitro by a classical rosette assay using IgG-coated sheep red blood cells. In RA patients, the percentage of monocytes forming rosettes was significantly lower than in controls (34.4 +/- 20.4 versus 67.4 +/- 4.5%; P less than 0.001). The blockade observed was reversed by a prior trypsin treatment of RA monocytes, the percentage of recovery being correlated with the IgG plasma levels. Besides, IgG purified from the serum of four RA patients bound a mean of 7.3, 5.2, 1.6, and 1.6 times more than normal IgG did onto concanavalin A (Con A), peanut agglutinin (PNA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), respectively. Although similar amounts of 125I-labeled normal and RA IgG were bound to normal monocytes, RA IgG inhibited more efficiently than normal IgG the Fc-R function of normal monocytes, for all concentrations tested (10 to 100 micrograms/100 microliters). A prior treatment of RA IgG by alpha-mannosidase, but not by beta-galactosidase, significantly reduced their inhibitory properties. The incubation of monocytes with D-mannose or mannan reduced their capacity to form rosettes. The percentage of monocytes forming rosettes in the presence of both mannan and normal IgG was significantly lower than that measured in the presence of normal IgG only. On the contrary, the rosetting capacity of monocytes in the presence of both RA IgG and mannan was the same as that calculated in the presence of RA IgG only. The inhibitory effect of RA IgG was not related to their abnormal circular dichroism. Our data suggest that the greater ability of RA IgG to block the Fc-R function of monocytes probably depends on the presence of a greater number of accessible mannosyl residues on the glycosidic side chains located in the Fc domain of the molecules.

Kininogens of low and high molecular mass, highly purified from human plasma, were used to raise antisera in rabbits. To obtain immunologically intact tracers, we labeled the kininogens by the lodogen method, followed by double chromatography, first on Sephadex G50 and then on Sephadex G200 for high-molecular-mass kininogen or on G100 for low-molecular-mass kininogen. Both assays are sensitive, accurate, and reproducible. On an equimolar basis, the high-molecular-mass kininogen cross reacted completely in the determination of low-molecular-mass analyte. Moreover, the radioimmunoassay for the former was highly specific. After optimizing the time and temperature of incubation to provide rapid and reliable results, we determined 95% reference intervals from a large sample of healthy subjects (250 men, 200 women): 109-272 and 69-116 mg/L for the low- and high-molecular-mass kininogens, respectively.

A non radioactive method for probing RNA or DNA on dot and Northern blots using a synthetic oligonucleotide with bromodeoxyuridine free ends is described. The present experiment was carried out with human testis and placental RNA's. The probe was the 21 base long sequence coding for the amino acids 18 to 24 of the insulin-like growth factor I (IGF-I) with two bromodeoxyuridine dinucleotides added at the 5' and 3' ends. The probe was detected with a monoclonal antibody against bromodeoxyuridine and immunogold silver staining (IGSS). Our method was compared to the peroxydase (HRP) revelation of the same probe. The results obtained show a lower background with IGSS than with HRP revelation. A sensitivity similar to that of 32P labelling was found with the advantages of an increase in the rapidity of the procedure (24 hours instead of 9 days exposure) and the absence of handling radioactive substances. Moreover, as the monoclonal antibody against BrdU detects single stranded DNA only, the use of BrdU free ends-labelled oligonucleotide allows the development of the revelation procedure without any previous denaturation of the hybrid. This particular point is an indisputable advantage for detecting hybridization in situ.

The amino acid sequence of the porcine beta subunit has been established by studies of peptides isolated after tryptic, thermolytic and staphylococcal protease treatments of the reduced and carboxymethylated chain. The primary structure of the amino-terminal region of the molecule has been solved by automatic sequencing of the reduced and tritium-labeled carboxymethylated subunit. The amino acid sequence of porcine follitropin beta subunit differs from that of its human counterpart by several amino acid replacements, deletion or addition of one or several residues. The porcine chain appears shorter at both its amino and carboxy-terminal ends. The chemical evolution of follitropin is briefly considered and compared to these of thyrotropin and lutropin.

Normal human placenta secretes within maternal compartment a pregnancy associated protein, placental growth hormone (PGH). This entity, agonist of pituitary GH, appears responsible for the elevated IGF I blood levels observed in the mother during pregnancy, while pituitary GH is no longer secreted. PGH could thus play a significant role in the anabolic processes of pregnancy. The biochemical mechanism responsible for PGH production is the expression of the GH-V gene at the placental level. This has been demonstrated by the positive probing of GH-V mRNA in this tissue, by the establishment of the restriction map and sequence of its cDNA as well as by the NH2-terminal sequence determination of both 22 and 25K PGH forms. The positive aspect of this function for human reproduction gains weight in that a placenta deleted for the CS-A, GH-V and CS-B gene expresses alternative genes coding for protein similar or related to pituitary GH or hPL, respectively.

Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.

The properties of porcine follitropin and its subunits which have not yet been characterized are presented. The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P-1 preparation. Its contamination by lutropin and thyrotropin amounted to 1 and 0.5 percent by weight respectively, as measured by radioimmunoassay. The alpha and beta subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography. The amino acid composition of porcine follitropin alpha subunit was found to be identical to that of alpha chain of porcine lutropin and thyrotropin. These porcine alpha chains differ, nevertheless, markedly in their carbohydrate composition particularly with respect to their mannose and galactose contents. The amino-terminal residue of the follitropin alpha subunit is threonyl. The carboxy-terminal end of the alpha chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin beta chain with glutamic acid at its carboxy-terminal end. Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively.

Quantitative data concerning the binding of 22000-mol.wt. human somatotropin and its 20000-mol.wt. variant are described using pregnant-rabbit liver and mammary-gland receptors. The purification and the complete chemical characterization of both human somatotropin and its 20000-mol.wt. variant is also presented. Contamination of the 20000-mol.wt.-variant preparation by 22000-mol.wt. hormone was found to be 0.5% by weight as measured in radioimmunoassay using monoclonal antibody. Labelling of human somatotropin and its 20000-mol.wt. variant using the Iodogen method is described as well as the characterization of the binding to pregnant-rabbit liver and mammary-gland receptor preparations. The maximum binding capacity of the 125I-labelled human somatotropin was between 50 and 60% to liver particulate receptor, whereas that of the 20000-mol.wt. variant was 30%. The specificity of binding of both forms to rabbit hepatic and mammary-gland receptor was found to be similar for both proteins in the same system. The affinity constants and capacity were respectively 0.7 X 10(10)M-1 and 815 fmol/mg of protein for human somatotropin and 0.6 X 10(10)M-1 and 1.250 fmol/mg of protein for the 20000-mol.wt. variant. These data suggest that both proteins behave as partial agonists to the receptors studied.