kDa Doublet Research Articles

FORSE-1: a positionally regulated epitope in the developing rat central nervous system

We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1-positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5-10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high-molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development.

Cellular and molecular characterization of a brain-enriched protein tyrosine phosphatase

Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64-66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64-66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64-66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.

Pharmacokinetics and bioactivity of intact versus truncated IGF-I during a 24-h infusion into lactating goats

The aim of this study was to compare the plasma concentration profile, mammary blood flow response and transfer into milk of intact IGF-I with that of its truncated analogue, des(1-3)IGF-I (des-IGF-I). Each peptide was infused for 24 h into the pudic artery supplying one mammary gland of lactating goats (n = 5). Concentrations of IGF-I in plasma (from the jugular vein) rose rapidly during infusion of IGF-I or des-IGF-I to reach 510 +/- 62 and 640 +/- 32 ng/ml (mean +/- S.E.M.) respectively, compared with 262 +/- 35 ng/ml after a similar infusion of saline. Ligand blotting analysis indicated a significant increase in the intensity of [125I]IGF-I binding to the 40-43 kDa doublet (binding protein-3 (BP-3), P < 0.01) and the band at 31 kDa (P < 0.05) during infusion of either IGF-I or des-IGF-I, as compared with saline. Furthermore des-IGF-I induced a significant increase in intensity of binding to the 35 and 24 kDa bands, but IGF-I did not. Whereas [125I]IGF-I was distributed between BP-3 and the other binding proteins, [125I]des-IGF-I bound exclusively to BP-3. Mammary blood flow (MBF) increased 48 +/- 6% after 12 h of infusion of des-IGF-I, compared with an increase of 22 +/- 6% during IGF-I. The difference in response was significant at P < 0.05. In addition, more IGF-I was secreted into the milk of the infused than the non-infused gland during either infusion of IGF-I or des-IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoblot studies of the IGF-related acid-labile subunit.

Insulin-like growth factors I and II (IGF-I and II) are present in serum primarily within a ternary complex consisting of IGF, IGF-binding protein-3 (IGF-3) and acid-labile subunit (ALS). Relatively little is known about ALS as compared to the other components of the complex. We report immunoblot studies of ALS using a new rabbit antiserum to human ALS1-34. The antiserum shows high specificity for ALS, labelling only the intact 82-88 kDa doublet in whole serum. Treatment with endoglycosidase-F leads to only a partial deglycosylation of ALS in whole serum, while purified ALS is reduced to M(r) approximately 58 kDa. Acidification of both whole serum and purified ALS leads to a complete loss of ALS ability to bind to cross-linked IGFBP-3:[125I]IGF-II tracer; however, immunoblot studies show no change in the apparent M(r) of the major ALS band. Immunoblot studies of human serum shows that intact ALS is decreased in growth-hormone (GH) deficiency, increases with GH treatment, is elevated in GH excess and is unchanged with IGF-I treatment. These data provide new information regarding the characteristics of ALS and demonstrate the research utility of a highly-specific antiserum for this protein.

Changes in the abundance of androgen receptor isotypes: effects of ligand treatment, glutamine-stretch variation, and mutation of putative phosphorylation sites.

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) migration pattern of wild-type and mutated human androgen receptors (ARs) expressed in COS-1 cells was analyzed. In the absence of hormone, the wild-type AR migrated as a closely spaced 110-112 kDa doublet. Alkaline phosphatase treatment resulted in a single 110 kDa band showing that the 112 kDa upshift reflects receptors phosphorylation. Deletion of the N-terminal amino acids 46-101 or 100-142 resulted in mutant ARs migrating as single protein bands. Three consensus phosphorylation sites in this region were substituted, and the resulting mutated proteins were analyzed. Two Ser-Pro-directed kinase consensus sites at positions Ser-80 and Ser-93 were both necessary for the AR 112 kDa upshift. Substitution of the putative casein kinase II Ser-118 site had no effect on the AR migration pattern. Surprisingly, deletion of the glutamine repeat, located directly N-terminal of the Ser-Pro sites, resulted also in an AR single form. Lengthening of the glutamine repeat caused an increase in the spacing between the two isotypes of the doublet, showing that the number of glutamine residues determines the extent of the upshift. Hormone treatment induced an extra isotype with an apparent molecular mass of 114 kDa, resulting in a 110-112-114 kDa AR triplet. The hormone-induced upshift was dependent on the Ser-80 consensus phosphorylation site. Mutations in the DNA binding domain caused a different distribution of receptor protein over the three AR isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a DNA-Binding Nuclear Autoantigen Mainly Associated with S Phase and G2 Cells

During analysis of autoimmune sera showing reactivity to nuclear antigens a serum producing a previously undescribed cell cycle-specific nuclear immunofluorescence pattern was observed. By indirect immunofluorescence the serum generated a densely speckled nucleoplasmic staining pattern of varying intensity with relative sparing of the nucleoli. The staining pattern could be abolished by treating cells with DNase but was not affected by RNase treatment. By flow cytometric dual parameter analysis the antigen expression was shown to increase daring S phase with a maximum in G2 cells, whereas G1 and M cells expressed low levels. The cell cycle-specific expression of the target antigen was similar in some aspects to the expression of cyclin A but clearly not identical. The serum recognized a 68/60 kDa doublet in immunoblotting of soluble MOLT-4 cell extract. Antibodies eluted separately from either of the two bands produced the cell cycle-specific staining pattern as well as the doublet in immunoblotting. DNA-cellulose was used for analysis of DNA-binding property and the higher band was found to be DNA-binding, whereas the lower band was not. In summary, this serum reacts with a DNA-binding and cell cycle-specific nuclear antigen which might be a novel protein involved in cell cycle progression.

The brain Kv1.1 potassium channel: in vitro and in vivo studies on subunit assembly and posttranslational processing

While combined cloning, mutagenesis, and electrophysiological techniques have provided great insight into K+ channel structure/function relationships, little is known about K+ channel biosynthesis. To examine K+ channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of 35S-met-labeled channels from in vitro translations and transfected mouse L-cells. When Kv1.1 and Kv1.4 were cotranslated in vitro, isoform-specific antisera copurified both proteins even at early time points, suggesting rapid subunit assembly. The non-Shaker Kv2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells transfected with Kv1.1 cDNA yielded 1000-4000 functional surface channels, and immune purification from Kv1.1 cells with Kv1.1 antisera produced a 57-59 kDa doublet on SDS-PAGE but not in sham-transfected cells. Immune purification of surface channels isolated both the 57 and 59 kDa proteins, suggesting cell surface channels are represented by two species. Pulse-chase metabolic labeling studies were consistent with a precursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase times, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mutation of the extracellular glycosylation site (N207) yielded two proteins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turnover, or function. Together these results suggest (1) heteromeric assembly of Shaker-like channels is cotranslational, and (2) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.

Fibroblast Growth Factor Regulates Insulin-like Growth Factor-Binding Protein Production by Vascular Smooth Muscle Cells

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

A site-directed antibody recognizes a component of the ouabain-binding domain of the alpha 1 subunit of rat Na+,K(+)-ATPase.

Antibodies (AbE1) were raised against an oligopeptide derived from a component of the ouabain-binding site of the alpha 1 subunit of rat Na+,K(+)-ATPase. Preincubation of partially purified Na+,K(+)-ATPase from rat kidney with 1 microM AbE1 partially inhibited the enzyme and reduced its sensitivity to 1 mM ouabain. Antibodies against an unrelated protein were ineffectual. Unexpectedly, the masses of the proteins detected on immunoblots of rat kidney by AbE1, a 160 kilodalton (kDa) doublet, was greater than the 97 kDa of the alpha 1 subunit. In lysates prepared with hot (85 degrees C) buffer constituted according to Laemmli's formulation for sample buffer (buffer L, a low chaotropic buffer), AbE1 always bound to the 160-kDa doublet. In contrast, AbE1 did not bind any proteins on blots of lysates that were prepared with buffer that contained greater concentrations of detergent and reducing agent (Laemmli's "homogenization" buffer, buffer H, a high chaotropic buffer), regardless of temperature. However, the presence of alpha 1 in lysates produced in homogenization buffer was confirmed by the presence of 97-kDa bands that were detected with two additional site-directed antibodies that recognize the alpha 1 subunit. When homogenates prepared with sample buffer L were examined, one of these antibodies bound to bands of 160 kDa, indicating that alpha 1 is a component of the bands bound by AbE1. It is concluded that AbE1 recognizes a component of the ouabain-binding site of Na+,K(+)-ATPase, but only under conditions in which the alpha 1 subunit was associated into a higher molecular mass complex.

Insulin-like growth factor binding proteins in the human adrenal gland

Insulin-like growth factors (IGFs) are thought to be important regulators of adrenocortical growth and steroidogenesis. IGFs are usually complexed with a family of specific IGF-binding proteins (IGFBPs) in serum, other body fluids, and in conditioned media of a variety of cell types. IGFBPs may either inhibit or potentiate the effects of IGFs. In the present study we have investigated the gene expression of the IGFBPs and IGF receptors in human fetal (HFA) and adult (HAA) adrenals. Northern blotting and/or reverse transcription polymerase chain reaction (RT-PCR) methods were used. IGFBP secretion into the cell culture medium was studied in primary cell cultures by Western ligand blotting and by radioimmunoassays. IGFBP-1 mRNA expression was low in adrenals: Northern blots were negative, but RT-PCR revealed IGFBP-1 mRNA in HFA. IGFBP-2 mRNA was equally expressed in both HFA and HAA with no differences in signal intensities by Northern blotting. IGFBP-3 mRNA was detected in HFA but not in HAA by Northern blotting. IGFBP-4 mRNA was expressed equally in both HFA and HAA. IGFBP-5 and -6 mRNA expression was more abundant in HAA than in HFA. IGF-I and type I and type II IGF receptor mRNAs were equally expressed in both HFA and HAA. 12- O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase regulator, upregulated IGFBP-1 in HFA cultures as determined by RIA, but ACTH was without effect. IGFBP-2 was not regulated by TPA or ACTH neither at protein nor at mRNA level. IGFBP-3 was downregulated by TPA both at protein and mRNA levels, but it was not affected by ACTH. In Western ligand blots of conditioned media from HFA cultures, we detected multiple bands. The best visualized band was a 47-44 kDa doublet, which most obviously represents IGFBP-3. Our results together with earlier findings show that all the components of the IGF system are expressed and produced locally in the human adrenal gland. There are also marked differences in the expression pattern of IGF-II and IGFBPs between fetal and adult glands. IGFBPs may have important autocrine or paracrine roles in the regulation of IGF functions in the adrenal gland.

Evidence that limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) occurs in the normal state outside of the bloodstream.

In serum and other biological fluids, IGF-I and IGF-II are reversibly bound to six molecular species of specific binding proteins (IGFBP-1 to -6) which regulate their transport to target cells as well as their biological activities. Most of the IGFs in adult serum are bound to IGFBP-3 and associated with a 85 kDa subunit to form 150 kDa ternary complexes, very few of which cross the capillary barrier. In earlier studies we showed that during pregnancy one or more serine proteinases are responsible for limited proteolysis of IGFBP-3 in the serum. This may result in easier dissociation of the IGFs and hence an increase in their availability. In the present work, the results of Western blot analyses of the IGFBPs, using either labelled IGF or a polyclonal anti-IGFBP-3 antibody, have demonstrated that IGFBP-3 proteolysis occurs in the normal state. In all the adults investigated, serum contained both the 42-39 kDa doublet, characteristic of intact IGFBP-3, and its major degradation fragment of 30 kDa. The fragment was also found in lymph, in addition to smaller fragments of 21.5, 20 and 16 kDa which are the same sizes as those seen in pregnancy serum. Comparisons of lymph (which reflects the interstitium) and serum from the same subjects showed greater proportions of IGFBP-3 proteolysed in lymph than in serum and incubations with [125I]IGFBP-3 showed almost 8-fold higher proteolytic activity in lymph than in serum where it was minimal. These findings suggest that the initial sites of proteolysis are in the tissues. Like that in pregnancy serum, the activity was calcium-dependent and inhibited by aprotinin. The results of this study fit the hypothesis that limited proteolysis of IGFBP-3 may be an essential mechanism in controlling the bioavailability of the IGFs, both at the cellular level and, in the blood, from the 150 kDa complexes which constitute the circulating reserves of IGF.

Active cdc2 genes and cell cycle phase‐specific cdc2‐related kinase complexes in hormone‐stimulated alfalfa cells

Hormones as auxins and cytokinins trigger the division cycle in differentiated plant cells and are required for the maintenance of proliferation in cultured cells in vitro. Northern analysis showed that the expression pattern of alfalfa cdc2 genes is significantly different in cells of primary explants exposed to hormone treatment and of rapidly cycling suspension culture. Transcription of at least one of the cdc2 genes is activated by hormones in cultured leaf mesophyll protoplasts or in root tissues treated with auxins and cytokinins. In a suspension culture of alfalfa cells, cdc2 transcripts are at a constitutively high level, irrespective of actual cell cycle phase. In addition to the transcriptional control of cdc2 genes, in hormone‐induced cells cdc2‐related kinase complexes were identified as potential phase‐specific components of post‐transcriptional regulation of the cell cycle. The known specific interaction between eukaryotic p34cdc2 protein complexes and the yeast p13suc1 protein was exploited for purification of cell cycle regulatory protein kinases from alfalfa. Alfalfa suspension cells were synchronized either for G1 phase by double‐phosphate‐starvation or for S phase by hydroxyurea treatment. The p13suc1‐Sepharose affinity matrix bound two cdc2 protein‐related complexes, one with increased histone H1 kinase activity in S, the other in G2/M phase. The complex from S phase cells showed higher kinase activity than the G2/M phase complex. Immunoblotting of p13suc1‐Sepharose‐bound protein complex showed the presence of a 33–34 kDa doublet that is recognized by anti‐PSTAIR antibodies and the co‐purification of two proteins (apparent molecular mass 65 and 42 kDa) cross‐reacting with human cyclin A antibodies. Immunoprecipitation with these cyclin A antibodies allowed the detection of a third cdc2‐related kinase complex that appeared during G1/S and early S phases of the cell cycle and phosphorylated histone H1. The results suggest that a multi‐component regulatory system is in control of the cell cycle in plants, which includes a hormone‐activated transcriptional control of cdc2 genes and phase‐specific cdc2‐related kinase complexes.

Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and beta 1 integrin receptors.

To learn about the effects of tension on fibroblast function, we have been studying initial cellular responses to stress-relaxation. Human foreskin fibroblasts were cultured in anchored collagen matrices for 2 days, during which time mechanical stress developed. Subsequently, the matrices were dislodged; thereby allowing stress to dissipate. Within 5 min after initiating stress-relaxation, fibroblasts retracted their pseudopodia. At this time, we observed the disappearance of cellular stress fibers and the formation of actin clusters along the cell margins. The actin was found to be located inside 200 nm diameter vesicles that were budding from the cell surface. Vesicles isolated from the matrix after stress-relaxation contained prominent 24 kDa, 36 kDa (doublet), 45 kDa, and 135 kDa polypeptides. The 45 kDa polypeptide was the major component in the Triton-insoluble vesicle fraction and appeared to be actin. The 36 kDa (doublet) polypeptide, which was found predominantly in the Triton-soluble vesicle fraction, was identified as annexin II. Vesicles also contained annexin VI and beta 1 integrin receptors but not tubulin, vimentin, vinculin or annexin I. The results suggest that stress-relaxation of fibroblasts induces a novel ectocytotic process involving transient budding of intact, plasma membrane vesicles from the cell cortex. On the basis of their morphological and biochemical features, these vesicles may be analogous to the 'matrix vesicles' released by chondrocytes and could play a role in extracellular matrix remodeling after wound contraction.

INTERLEUKIN-1 DIFFERENTIALLY STIMULATES INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 RELEASE FROM HUMAN ARTICULAR CHONDROCYTES

Articular chondrocytes create and maintain articular cartilage and their growth and metabolism are tightly regulated. The insulin-like growth factors (IGFs), and hence the IGF binding proteins (IGFBPs), play a significant, but unclear, role in this regulation. We examined the articular chondrocyte release of IGFBPs under conditions that simulate inflammation. Human chondrocytes were isolated from articular cartilage obtained post-mortem from a three year old female. The cells were grown in serum free conditions in high density monolayers. Interleukin-1 (IL-1), a central mediator of the inflammatory response, was added to a final concentration of 30 ng/ml and cultures incubated for 4, 12, 24, or 48 hours. Conditioned media were analyzed for IGFBPs by Western ligand blotting, autoradiography, and laser densitometry. Conditioned media from unstimulated chondrocytes contained IGFBPs at 22, 27, 38, and 41 kDa and their concentrations increased linearly over 48 hours. The 38/41 kDa doublet has been shown in serum to be IGFBP-3. Conditioned media from the IL-1 stimulated chondrocytes contained the same forms of IGFBP, plus a 34 kDa IGFBP at 48 hours. The rate of accumulation of the IGFBP-3 doublet from the IL-1 treated chondrocytes was 3.1±0.4 (SE) times the rate from the unstimulated chondrocytes (P<0.05). The rates of accumulation of the 22 and 27 kDa bands were not different. IGFBPs are produced by human chondrocytes in vitro, and IL-1 differentially increases the rate of accumulation of IGFBP-3. IGF-1 has profound effects on articular chondrocytes in vitro, and may play a role in articular cartilage disease. The finding that chondrocytes increase the release of an IGFBP in response to the cytokine IL-1 supports this hypothesis.

Changes in insulin-like growth factor-I (IGF-I), IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles

Levels of insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) were studied in ovine follicular fluid and serum with a view to determining their respective endocrine and/or paracrine roles in ovarian folliculogenesis. Ovarian follicles of Ile-de-France ewes were dissected and measured individually, and their follicular fluid collected. Follicles were classified according to size and degree of atresia, assessed on the basis of microscopical examination of smears of granulosa cells. Follicular fluid and serum samples were assayed for IGF-I and IGF-II. Free IGF-binding activity was also determined, and the IGFBP profiles in serum and follicular fluid were examined by Western ligand blotting [44- to 42-kilodalton (kDa) doublet and 35-kDa, 28.5- to 32-kDa, and 24-kDa bands], followed by densitometric analysis of the autoradiographs. Finally, the effects of follicular fluid IGFBPs on granulosa cell responses to IGF-I were studied in vitro. The size and atretic stage of the follicles had little influence on the IGF-I concentrations in follicular fluid, but IGF-II concentrations were approximately 1.5 times higher in small than in large follicles (P < 0.01). IGF-I levels were lower in fluid from large normal (highly vascularized) follicles than in serum (P < 0.01). Follicular fluid and serum IGF-I levels were positively correlated (r = 0.55; P < 0.05). No significant difference was found between follicular fluid and serum IGF-II levels. Follicular growth was accompanied by a decrease in free IGF-binding activity (P < 0.0001), a slight increase in the intensity of the 44- to 42-kDa IGFBP doublet (P < 0.05), and a clear decrease in the intensities of the 35-kDa (P < 0.0001) and 24-kDa bands. By contrast, follicular atresia was characterized by a marked increase in free IGF-binding activity (P < 0.0001) and strongly increased intensities of the 35-kDa, 28.5- to 32-kDa, and 24-kDa bands (P < 0.0001). Low mol wt IGFBPs, particularly the 24-kDa species, were clearly more abundant in serum than in follicular fluid from large normal follicles. In vitro experiments showed IGF-I to be less active on granulosa cells in the presence of follicular fluid from atretic than from normal follicles. The action of the IGF-I analog [Gln3,Ala4,Tyr15,Leu16]IGF-I, which has a weak affinity for the IGFBPs, was, however, similar whether atretic or normal follicular fluid was tested.

Changes in insulin-like growth factor-I (IGF-I), IGF-II, and their binding proteins during growth and atresia of ovine ovarian follicles.

Levels of insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) were studied in ovine follicular fluid and serum with a view to determining their respective endocrine and/or paracrine roles in ovarian folliculogenesis. Ovarian follicles of Ile-de-France ewes were dissected and measured individually, and their follicular fluid collected. Follicles were classified according to size and degree of atresia, assessed on the basis of microscopical examination of smears of granulosa cells. Follicular fluid and serum samples were assayed for IGF-I and IGF-II. Free IGF-binding activity was also determined, and the IGFBP profiles in serum and follicular fluid were examined by Western ligand blotting [44- to 42-kilodalton (kDa) doublet and 35-kDa, 28.5- to 32-kDa, and 24-kDa bands], followed by densitometric analysis of the autoradiographs. Finally, the effects of follicular fluid IGFBPs on granulosa cell responses to IGF-I were studied in vitro. The size and atretic stage of the follicles had little influence on the IGF-I concentrations in follicular fluid, but IGF-II concentrations were approximately 1.5 times higher in small than in large follicles (P < 0.01). IGF-I levels were lower in fluid from large normal (highly vascularized) follicles than in serum (P < 0.01). Follicular fluid and serum IGF-I levels were positively correlated (r = 0.55; P < 0.05). No significant difference was found between follicular fluid and serum IGF-II levels. Follicular growth was accompanied by a decrease in free IGF-binding activity (P < 0.0001), a slight increase in the intensity of the 44- to 42-kDa IGFBP doublet (P < 0.05), and a clear decrease in the intensities of the 35-kDa (P < 0.0001) and 24-kDa bands. By contrast, follicular atresia was characterized by a marked increase in free IGF-binding activity (P < 0.0001) and strongly increased intensities of the 35-kDa, 28.5- to 32-kDa, and 24-kDa bands (P < 0.0001). Low mol wt IGFBPs, particularly the 24-kDa species, were clearly more abundant in serum than in follicular fluid from large normal follicles. In vitro experiments showed IGF-I to be less active on granulosa cells in the presence of follicular fluid from atretic than from normal follicles. The action of the IGF-I analog [Gln3,Ala4,Tyr15,Leu16]IGF-I, which has a weak affinity for the IGFBPs, was, however, similar whether atretic or normal follicular fluid was tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of developmentally-regulated nucleases in promastigotes and amastigotes of Leishmania mexicana

The parasitic protozoon Leishmania mexicana was examined for the presence of 3'-nucleotidase/nuclease using substrate SDS-PAGE. Two activities were detected: one with an apparent molecular mass of 40 kDa, and a doublet of 29/31 kDa. The two enzymes showed differences in their levels of expression in the two life-cycle stages of parasite examined: the 29/31 kDa doublet was detected at 60-fold higher levels in the pathogenic amastigote stage, but was also expressed by promastigotes, whereas the 40 kDa activity was only detected in promastigotes. Both were capable of hydrolysing a variety of substrates including 3'-AMP and poly(A). However, the 29/31 kDa form showed a broader, unique substrate specificity in that it was also capable of digesting double-stranded RNA and DNA.

Regulation of insulin-like growth factor binding protein production by human luteinizing granulosa cells cultured in defined medium.

The production of insulin-like growth factor (IGF) binding proteins (IGFBPs) by human luteinizing granulosa cells obtained at oocyte harvest for in vitro fertilization was studied in defined medium. Three species of IGFBP were noted on Western ligand blotting of conditioned medium. A 31-33 kilodalton (kDa) doublet identified as IGFBP-2 by immunoprecipitation was consistently seen and was shown by metabolic labeling to be synthesized de novo. IGFBP-2 production was inhibited by hCG (100 ng/mL) to 32 +/- 8% of levels in control cultures; a similar inhibition was seen with dibutyryl cAMP and forskolin, but not with FSH. IGFBP-2 production was also inhibited by IGF-II and [Leu27]IGF-II (ED50 for both 3 ng/mL) but not by IGF-I at up to 30 ng/mL. In addition to IGFBP-2, a 35-45 kDa IGFBP identified by immunoprecipitation as IGFBP-3 was variably present on ligand blots of control conditioned medium and was consistently found in medium from cells treated with IGF-I or IGF-II (ED50 3 ng/mL) but not with insulin at 2 mg/mL. No de novo synthesis of IGFBP-3 was noted, suggesting that IGFBP-3 may be released from the cell surface by ligand. A 24-kDa IGFBP consistent with IGFBP-4 was also variably noted in basal cultures. Since IGFBPs are believed to sequester IGF peptides and inhibit their action on the granulosa, control of IGFBP production may be an important step in regulating gonadotropin and IGF action in the human granulosa cell.

Comparative Use of Immunological Methods &amp; Ligand Blotting of Insulin-like Growth Factor Binding Proteins in Serum and Other Biological Fluids

There is controversy as to which methodology most accurately detects the Insulin-like growth factor binding proteins (IGFBPs). We have compared immunological and ligand binding techniques to assess the IGFBPs in serum from non-pregnant women and women in late pregnancy, fetal cord serum and amniotic fluid. A specific radioimmunoassay for IGFBP-3 measured mean levels of 3.5 and 3.7μg/ml for non-pregnancy and pregnancy serum, respectively, 0.7μg/ml for fetal cord serum and 5.1μg/ml for amniotic fluid. In contrast, Western-ligand blot of fluid showed IGFBP-3 as a 40 kDa doublet in non-pregnancy serum and fetal cord serum, unlike pregnancy serum or amniotic fluid, where IGFBP-3 was undetectable by standard blot analysis. However, analysis of fluids at 25-50 times more volume showed that IGFBP-3 was detectable as a 40 kDa doublet in pregnancy serum and amniotic fluid. The molecular mass of immunoreactive IGFBP-3 was examined by Western-immunoblot, probed with antibody α IGFBP-31l. As in Westem-ligand blot, intact IGFBP-3 (40 kDa) was easily detected in non-pregnancy serum and fetal cord serum, but required greater volumes of sample for detection in pregnancy serum and amniotic fluid. In addition, smaller immunoreactive fragments were found in all fluids: a faint 27 and 22 kDa band was found in non-pregnancy serum and fetal cord serum, with more dominant bands of 31-27, 26, 25, 24 and 22 kDa in pregnancy serum and 31-26, 25, 22, 19-17 kDa in amniotic fluid. To address the obvious discrepancies between ligand and immunological analysis, samples were assessed for IGFBP-3 protease activity. Non-pregnancy serum proteolyzed 14% of the substrate, pregnancy serum 61%, fetal cord serum 33%, and amniotic fluid 50-61%. Thus, fluids with apparently low IGFBP-3 by Western-ligand blot, but high IGFBP-3 by RIA, showed high levels of protease activity. Previously, IGFBP-proteases have been shown to degrade IGFBP to smaller fragments with a reduced affinity for IGF. We conclude that the discrepancy between ligand binding techniques and immunological techniques can be explained, at least in part, by the presence of IGFBP-protease activity.

Proteins of the honeybee ( Apis mellifera L.) body surface and exocrine gland secretions

In this paper, soluble proteins are demonstrated on the surface of the honeybee cuticle. Polyacrylamide gel electrophoresis revealed an 80 kDa doublet on workers, queens, and drones showed a single band at a slightly higher molecular weight. Several additional queen- or drone-specific proteins were observed. The presence of soluble cuticular surface proteins may be a general phenomenon because they were also present on insects of two other orders, Orthoptera and Hemiptera. Exocrine gland secretions from the Nasanov glands of workers and the tarsal glands of both female castes and drones also contained soluble proteins. Proteins of tarsal gland secretions may contribute to the milieu of surface proteins because the electrophoretic profiles of cuticular proteins and tarsal secretions were similar. In contrast, the electrophoretic profile of proteins from Nasanov gland secretions was distinct from the profile of the tarsal gland secretion. Proteins on the cuticular surface or in exocrine gland secretions could also affect social interactions. Surface cuticular proteins and those associated with exocrine glands may function in pheromone degradation, as slow release substances, or as caste- or sex-specific modulators of pheromone activity.