Palaeoclimate evidence of vulnerable permafrost during times of low sea ice.

Climate change in the Arctic is occurring rapidly, and projections suggest the complete loss of summer sea ice by the middle of this century1. The sensitivity of permanently frozen ground (permafrost) in the Northern Hemisphere to warming is less clear, and its long-term trends are harder to monitor than those of sea ice. Here we use palaeoclimate data to show that Siberian permafrost is robust to warming when Arctic sea ice is present, but vulnerable when it is absent. Uranium-lead chronology of carbonate deposits (speleothems) in a Siberian cave located at the southern edge of continuous permafrost reveals periods in which the overlying ground was not permanently frozen. The speleothem record starts 1.5 million years ago (Ma), a time when greater equator-to-pole heat transport led to a warmer Northern Hemisphere2. The growth of the speleothems indicates that permafrost at the cave site was absent at that time, becoming more frequent from about 1.35 Ma, as the Northern Hemisphere cooled, and permanent after about 0.4 Ma. This history mirrors that of year-round sea ice in the Arctic Ocean, which was largely absent before about 0.4 Ma (ref. 3), but continuously present since that date. The robustness of permafrost when sea ice is present, as well as the increased permafrost vulnerability when sea ice is absent, can be explained by changes in both heat and moisture transport. Reduced sea ice may contribute to warming of Arctic air4-6, which can lead to warming far inland7. Open Arctic waters also increase the source of moisture and increase autumn snowfall over Siberia, insulating the ground from low winter temperatures8-10. These processes explain the relationship between an ice-free Arctic and permafrost thawing before 0.4 Ma. If these processes continue during modern climate change, future loss of summer Arctic sea ice will accelerate the thawing of Siberian permafrost.

Requirement for activin A and transforming growth factor--beta 1 pro-regions in homodimer assembly.

Many proteins are initially synthesized as part of a large precursor. The role of the pro-region in the biosynthesis of transforming growth factor--beta 1 (TGF-beta 1) and activin A, two structurally related disulfide-linked homodimers synthesized as large precursors, was studied. Vectors that expressed either the pro-region or the mature regions of these molecules were used in complementation experiments, only when the pro-region was coexpressed with the mature region did intracellular dimerization and secretion of biologically active homodimers occur. The pro-regions of activin A and TGF-beta 1, therefore, aid the folding, disulfide bond formation, and export of their respective homodimers.

Secretion of activin by interstitial cells in the testis.

Activin, a dimer formed by the beta subunits of inhibin, has an effect that is opposite to that of inhibin in a number of biological systems. Which cell types secrete activin in vivo is not known. TM3 cells, a Leydig-derived cell line, contained messenger RNAs that hybridized with human beta A and beta B complementary DNA probes and were similar in size to the porcine messenger RNA for the beta subunits of inhibin. No hybridization to the inhibin alpha subunit was detectable in the TM3 cells. Conditioned medium from TM3 cells and from primary cultures of rat and porcine interstitial cells stimulated the release of follicle-stimulating hormone in a pituitary cell culture assay. It is likely that, in the testis, the Leydig cells secrete activin and the Sertoli cells produce inhibin, or a combination of both.

The hypogonadal mouse: reproductive functions restored by gene therapy.

The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.

A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse.

Hereditary hypogonadism in the hypogonadal (hpg) mouse is caused by a deletional mutation of at least 33.5 kilobases encompassing the distal half of the gene for the common biosynthetic precursor of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP). The partially deleted gene is transcriptionally active as revealed by in situ hybridization histochemistry of hpg hypothalamic tissue sections, but immunocytochemical analysis failed to show the presence of antigen corresponding to any part of the precursor protein.

Rational design of vector and antibiotic peptides using solid-state NMR.

The application of 2H solid-state NMR in determining structure activity relationships and mechanism of action of membrane active peptides is discussed. The enhancement of the disruption of anionic lipids in the membrane by new lead compounds is shown to be a key determinant of both DNA vector and antimicrobial activity.

Purification and characterization of recombinant human activin B.

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Functional analysis of the cysteine residues of activin A.

Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A.

Recombinant expression of human alpha- and beta A-inhibin subunit cDNAs in mammalian 293 cells results in the secretion of 20-53K free alpha-subunit-derived products, 30-105K alpha beta A-inhibin dimers, and 24-110K beta A-activin dimers. The present study verifies that the wide variation in the size of these products is due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked glycosylation site at amino acid number 302 in the alpha C-subunit. The identity of each of these products was established by mutagenesis of proteolytic processing sites and N-linked glycosylation sites, combined with the analysis of transfection products by immunoprecipitation and one- and two-dimensional SDS-PAGE (SDS/SDS-beta-ME). Transient expression of processing site mutants of the alpha- and beta A-subunits in 293 cells was used to generate microgram quantities of noncleavable 55K and 65K inhibin dimers, and noncleavable 110K activin A dimers. The 55K and 65K inhibin A forms were purified and found to be fully biologically active in a rat pituitary cell bioassay. The 110K high molecular weight (HMW) form of human activin A failed to show any FSH-releasing activity in the pituitary assay. Since radioactively labeled 55K and 65K inhibin A and 110K activin A remained intact after incubation with rat pituitary cells for 72 h, there appears to be no conversion of these dimers to lower molecular weight forms by proteolytic cleavage at additional sites. These results show for the first time that 55K and 65K inhibit A are intrinsically biologically active and do not require cleavage to the 32K form for activation. In contrast, cleavage of the 110K activin A precursor to the 24K form would appear to be necessary for activity.

Recombinant expression and characterization of human activin A.

Activin, which stimulates the secretion of FSH from anterior pituitary cells, is a dimer of the beta-subunits of inhibin. Two species of activin (A and AB) have been purified from ovarian follicular fluid and characterized. We have been able to biosynthetically produce recombinant human activin A by constructing stable cell lines expressing the mRNA for the beta A subunit of human inhibin. These cell lines secreted a 24 kilodalton beta A dimer which stimulated FSH secretion in cultured pituitary cells. The ability of this protein to stimulate FSH secretion was sensitive to reduction of disulfide bonds, exhibited a slow onset of action, and was blocked by actinomycin D. In addition, recombinant activin A stimulated hemoglobin accumulation in K562 cells. These data show that recombinant activin A has the biochemical properties and biological activities that have been ascribed to native activin A. In addition, these results provide an independent confirmation, and thereby a final proof, of the structure and function of activin.

Activin B: precursor sequences, genomic structure and in vitro activities.

We report here the complete amino acid sequence of the human inhibin beta B-subunit as deduced from the sequence of cDNA and genomic clones. The primary translation product of the beta B mRNA predicts a protein of 407 amino acids, containing a prepro region of 292 amino acids separated by basic amino acids from the mature C-terminal 115 amino acids. Mammalian tissue culture cells transfected with a beta B-subunit expression plasmid secreted an activin B homodimer of approximately 22K mol wt. Coexpression of the beta A- and beta B-subunit mRNAs resulted in the secretion of the three forms of activin, A, AB, and B. Purified activin B was shown to elicit FSH release in an in vitro pituitary assay and trigger the accumulation of hemoglobin in K562 cells. The potency of activin B in both of these assays (ED50 approximately 2 ng/ml) was indistinguishable from that observed for activin A.

Localization of inhibin/activin subunit mRNAs within the primate ovary.

In order to gain further understanding of the physiology of inhibin and activin in the primate, the expression of inhibin/activin subunit mRNAs in the monkey ovary was examined by in situ hybridization. Granulosa cells of small antral follicles were found to express mRNA for the beta B subunit, which decreased to undetectable levels in dominant follicles. In contrast, expression of alpha and beta A subunit mRNAs was detected in granulosa cells of dominant follicles and in corpora lutea, but not in small antral follicles. These results indicate that the expression of the beta A and beta B subunits is differentially regulated during the growth and development of ovarian follicles in the monkey.

Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor.

The human osteoinductive proteins BMP-2a and BMP-2b have been cloned and expressed in mammalian cells. In order to improve expression levels we examined the role of the proregion in assembly and export. Use of the BMP-2a proregion combined with the mature region of BMP-2b leads to dramatically improved expression of mature BMP-2b. Mature BMP-2b has been purified to near homogeneity from the BMP-2a/2b hybrid, and its structural properties and biological activity determined. Recombinant mature BMP-2b homodimer elicits bone formation in vivo.

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins.

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.

Effects in vivo of recombinant human inhibin on myelopoiesis in mice.

Inhibin, a protein dimer, has been implicated in negative regulation of human erythropoiesis in vitro. In this study, purified recombinant human (rhu) inhibin was assessed for its effect in vivo on the proliferation of hematopoietic progenitor cells in C3H/HeJ mice. Administration of single doses of inhibin i.v. to mice resulted 24 hrs later in significant decreases in cell cycling status of marrow and splenic granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitors. While no apparent effect was observed on marrow cellularity or on absolute numbers of marrow CFU-GM and BFU-E, inhibin significantly reduced absolute numbers of marrow CFU-GEMM, spleen nucleated cellularity and also absolute numbers of CFU-GM, BFU-E and CFU-GEMM in the spleen. The results demonstrate in vivo myelosuppressive effects for inhibin and demonstrate that effects in vivo are not restricted to erythropoiesis.

Multiple actions of recombinant activin-A in vivo.

Activin stimulates the secretion of FSH from cultured pituitary cells and enhances the differentiation of erythroid progenitors in vitro. The role of activin in the physiological regulation of either process, however, is unknown. We report here that systemic administration of recombinant human activin-A to immature female rats caused a marked increase in serum FSH levels. In addition, in ovariectomized estrogen-treated rats recombinant human activin-A induced a small but statistically significant increase in the circulating concentrations of red blood cells and hemoglobin. These data demonstrate the efficacy of activin in vivo, supporting the hypothesis that this protein is an important regulator of gonadotropic and erythroid function.

Recombinant human activin-A stimulates basal FSH and GnRH-stimulated FSH and LH release in the adult male macaque, Macaca fascicularis.

Activin-A is a homodimer of the beta A inhibin subunit that stimulates FSH secretion by pituitary cells in vitro; however, the physiological relevance of this effect is unknown. We have examined whether recombinant human activin-A (activin-A; 80 micrograms/kg/day iv infusion for 50.5 h) has in vivo bioactivity in the adult male macaque (n = 5). Serum FSH and LH bioactivity and serum testosterone (T) levels were measured on 2 control days and after 24 and 48 h of activin-A administration. Basal FSH levels increased significantly (p less than 0.05) by 17% at 24 h and 82% at 48 h during activin-A administration. No changes in basal LH or T levels were seen. The FSH and LH responses to GnRH (5 micrograms/kg, iv bolus) increased significantly (p less than 0.05) by 117% and 55% after 48 h of activin-A, respectively. A small (16%), but statistically significant (p less than 0.05), increase in the T response to the GnRH challenge was also noted. These data are preliminary evidence in support of a physiological role for activin-A in the control of gonadotropin secretion in the male primate.

Evidence for an autocrine role of activin B within rat anterior pituitary cultures.

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.

Immunoreactive inhibin alpha-subunit in human serum: implications for radioimmunoassay.

The specificity of the most widely used heterologous RIA for human serum inhibition was examined with regard to its cross-reactivity with the alpha-subunit of inhibin. Using a mixture of recombinant alpha inhibin proteins, including precursor and mature forms, the epitopic specificity of this antibody was localized entirely to the alpha-subunit. Furthermore, varying degrees of alpha inhibin immunoactivity were demonstrated in serum from normal subjects and patients with various reproductive disorders by both Western blot and RIA. These results demonstrate that data obtained using this RIA should be interpreted with caution as to the degree to which they reflect the presence of intact dimeric inhibin vs. alpha inhibin proteins in the circulation.

Effect of recombinant activin on androgen synthesis in cultured human thecal cells.

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.