The effect of entering drug treatment on involvement in HIV-related risk behaviors.

The research described here is based on a sample of 8,241 out-of-drug-treatment users of injected drugs and/or crack, aged 18 or older, recruited from 22 sites across the United States and Puerto Rico. The study divided respondents into three groups-(a) cocaine or crack users who did not also use heroin or speedball (cocaine-only users), (b) heroin injectors who did not also use cocaine or crack or speedball (heroin injectors), and (c) users of cocaine or crack and injected heroin or speedball (dual users)--and compared the efficacy of entering drug treatment for these groups' involvement in HIV-related risk behaviors. The study found that entry into treatment corresponded to greater reductions in substance abusers' frequency of drug use and involvement in risky injection practices compared to those observed in people who did not enter treatment between their baseline and 6-month follow-up interviews. Entry into drug treatment was also associated with reductions in the practice of risky sexual behaviors, but these reductions were less substantial and less consistent than those noted for drug use and injection risk behaviors.

Positive selection of growth-inhibitory genes.

We have isolated a limited set of cDNAs that limit cell proliferation using a unique assay based on the dilution of a lipophilic fluorescent dye as transfected cells divide. The identification of growth-inhibitory factors has been limited by the lack of a strong assay for growth inhibitors. A growth-inhibited cell does not grow and so is at a selective disadvantage in vitro when compared with any growing cell. Several assays have been used to screen for growth-inhibitory genes; however, these approaches are either very difficult to implement, leaky, or not comprehensive. We have developed an assay that selects for cDNAs capable of inhibiting proliferation in which cells are nonspecifically labeled with a lipophilic fluorescent dye, PKH-2, and subsequently transfected with a cDNA library made from growth-inhibited cells. With each cell division, the amount of dye per cell is reduced by one-half. Over time, growth-inhibited cells will retain more dye per cell relative to actively growing cells. The population is then analyzed by fluorescence-activated cell sorting, and the brightest cells in the population are isolated. This assay has allowed us to select pools of cDNAs enriched for growth-inhibitory activities and may provide a general method for identifying growth-inhibitory genes active in varying biological contexts. We report here the successful application of the dye retention assay to the selection of cDNAs that inhibit epithelial cell proliferation.

Identification of growth inhibited cells by retention of a lipophilic fluorescent dye.

Cellular proliferation is regulated in both positive and negative ways. However, direct selection for growth inhibitory control elements is limited by the difficulty in identifying a growth inhibited cell against a background of cells which are proliferating. This study describes a positive selection technique for growth inhibited cells. This method is based on the retention of a lipophilic fluorescent dye which nonspecifically labels plasma membranes and distributes between daughter cells with membrane lipid as cells proliferate. Characterization of this assay is described using an epithelial cell line which is growth inhibited in response to transforming growth factor beta (TGF-beta) and dexamethasone and several mutant clones of that line which lack responsiveness to TGF-beta. Retention of dye in response to the growth inhibitors is proportional to the inhibition of thymidine incorporation of those cells. Mixing experiments were also carried out in which G418 resistant TGF-beta responsive epithelial cells were mixed with TGF-beta nonresponsive mutants. The mixture was labeled with PKH-2 and exposed to TGF-beta for 3 days. Subsequently, consecutive fractions of cells sorted on the basis of fluorescence intensity were selected in G418, and the TGF-beta responsive epithelial cells were found predominantly in the most fluorescent cells in the population. This method provides a positive selection for growth inhibited cells which may, in combination with classical gene transfer techniques, provide a way to select for growth inhibitory genes in a manner analogous to the focus forming assay selection for oncogenes.

Responsiveness to transforming growth factor-beta (TGF-beta) restored by genetic complementation between cells defective in TGF-beta receptors I and II.

Selection of mutant Mv1Lu mink lung epithelial cells resistant to growth inhibition by transforming growth factor-beta (TGF-beta) has led to the isolation of cell clones with distinct alterations in type I and II TGF-beta receptors. Certain mutant clones present a decreased number or complete loss of detectable type I receptor. Other clones show a loss and/or altered electrophoretic mobility of the type II receptor, with concomitant loss of the type I receptor. Using somatic cell hybridization analysis we demonstrate the recessive nature of these mutants with respect to the wild-type phenotype and define various mutant complementation groups. Among these, hybrids between cells that express only type II receptor (R mutants) and cells that express neither receptor type (DRa mutants) rescue wild-type expression of type I receptors. Moreover, these hybrids regain full responsiveness to TGF-beta 1, as measured by inhibition of DNA synthesis as well as stimulation of fibronectin and plasminogen activator inhibitor-1 production. These results provide evidence for an interaction between TGF-beta receptor components I and II and show that, in Mv1Lu cells, expression of both receptor types is required for mediation of biological responses to TGF-beta 1.

Transforming growth factor-beta receptors and binding proteoglycans.

Transforming growth factors-beta (TGFs-beta) are representative of a superfamily whose members were first identified as regulators of morphogenesis and differentiation, and subsequently found to be structurally related. Other members of the family include the activins and inhibins, BMPs, MIS, the DPP-C gene product and Vg-1. When assayed by affinity-labelling techniques, TGFs-beta bind to three distinct cell surface proteins which are present on most cells. These proteins are all of relatively low abundance but bind TGFs-beta with affinities consistent with the biological potency of the factors. The Type I and Type II binding proteins are glycoproteins with estimated molecular weights of 53 and 73 x 10(3) Mr, respectively. They both bind TGF-beta 1 significantly better than TGF-beta 2. The Type I receptor has been identified as the receptor which mediates many of the responses of TGFs-beta, based on somatic cell genetic studies of epithelial cell mutants unresponsive to TGFs-beta. Betaglycan is the third binding protein present on many, but not all, cell types and is a large proteoglycan (approximately 280 x 10(3) Mr) with 100-120 x 10(3) Mr core proteins. A soluble form of this molecule is present in conditioned media of many cell lines and may be derived from the cell surface-associated molecule by cleavage of a small membrane anchor. Betaglycan binds TGF-beta 1 and TGF-beta 2 with similar affinity and this binding is to the core proteins, not the glycosaminoglycan side chains. This molecule may have a function in the localization and delivery or the clearance of activated TGFs-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor.

Cells whose proliferation is blocked by transforming growth factor-beta (TGF-beta) express three distinct surface glycoproteins of 53, 73, and 300 kDa that bind TGF-beta with high affinity, but whose function is unknown. We have isolated two classes of chemically-induced Mv1Lu epithelial cell mutants resistant to growth inhibition by TGF-beta. Class R mutants have selectively lost expression of the 53-kDa (type I) TGF-beta-binding protein. They have also lost the ability to respond to TGF-beta with elevated fibronectin expression and cell flattening. Class S mutants bind normally but do not respond to TGF-beta. TGF-beta-resistant mutants retain a contact inhibited, nontransformed phenotype. The properties of S mutants suggest that they are defective in the TGF-beta signal transduction mechanism, while the results with R mutants identify the type I TGF-beta-binding protein as the receptor involved in mediating TGF-beta actions on cell adhesion and proliferation.

Multiple type-beta transforming growth factors and their receptors.

Type beta transforming growth factors are a group of homologous structurally related polypeptides that act on a wide variety of cell types to alter their proliferative and phenotypic properties. TGF-beta s form a group within a larger family of polypeptides that control developmental processes in organisms from humans to Drosophila. We have found that at least three distinct forms of TGF-beta are present in mammalian tissues. We have identified a family of cell surface glycoproteins that bind TGF-beta s with high affinity and specificity. Examination of the interactions between individual forms of TGF-beta and the individual TGF-beta receptor species has illustrated a complex pattern of ligand-receptor associations. Occupancy of a particular receptor type by TGF-beta can be correlated to the dictation of specific effects on cell proliferation and cell differentiation.

Insulin inhibits specific norepinephrine uptake in neuronal cultures from rat brain.

Neuronal cells in primary culture have been demonstrated to possess specific insulin receptors (Boyd et al., J. Biol. Chem., 260 (1985) 15880-15884). Incubation of these cultures with insulin causes a dose-dependent inhibition of maprotiline-sensitive [3H]norepinephrine uptake. Maximum inhibition of 95% of maprotiline-sensitive norepinephrine uptake was observed at an insulin concentration of 167 nM with an ED50 of 30 nM. Competition-inhibition and Scatchard analysis of the insulin binding data suggested that maprotiline competed for high-affinity insulin receptors. These observations suggest that both insulin and maprotiline specifically inhibit neuronal norepinephrine uptake possibly involving insulin receptors.

Insulin is released from rat brain neuronal cells in culture.

Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than three-fold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization-induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions.

Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures.

We studied the structural and functional characteristics of insulin receptors from rat brain and liver from late gestation through adulthood as well as from cultured neuronal and glial cells from neonatal rats. Specific insulin binding was present on membrane preparations from brain and liver at all stages of development studied, with maximal binding in neonates greater than 19-day-old fetuses greater than adults for both brain and liver. Maximal specific binding to cultured neuronal and glial cell membranes was similar (6.2% vs. 7.1%, respectively). [125I]Iodoinsulin cross-linking to the insulin receptor demonstrated that the mol wt (Mr) of the brain alpha-subunit was less than that of the liver alpha-subunit at all stages. [125I]Iodoinsulin cross-linking also demonstrated that the glial cell alpha-subunit (Mr, 130,000) migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to a position intermediate between the liver (Mr, 135,000) and brain (Mr, 119,000), whereas the neuronal cell alpha-subunit (Mr, 118,000) comigrated with the brain alpha-subunit. In solubilized lectin-purified preparations from brain and liver during development as well as from neuronal and glial cells, insulin stimulated phosphorylation of the beta-subunit. The Mr of the brain beta-subunit, as determined by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was less than that of the liver beta-subunit. The neuronal cell beta-subunit comigrated with the brain beta-subunit while the glial cell beta-subunit migrated to a position intermediate between the brain and liver beta-subunit. Solubilized lectin-purified preparations from all tissues demonstrated insulin-stimulable phosphorylation of exogenous substrates. From these studies we conclude that 1) functional insulin receptors are present in the brain during development in the rat; and 2) the structural differences demonstrated between neuronal and glial cell and between brain and nonneuronal insulin receptors taken together with previously demonstrated functional differences of the insulin receptor on these tissues suggest a unique function for insulin receptors on neuronal tissues.

Insulin is a growth-promoter of the developing brain: possible implications for the infant of the diabetic mother.

The combination of diabetes mellitus and pregnancy was once associated with significantly increased morbidity and mortality for mother and baby. Advances in the obstetric management of the mother with diabetes and parallel advances in the management of the infant of the diabetic mother (IDM) have lowered the morbidity and mortality in many centers to near those for pregnancies in the non-diabetic woman. A significant increase still exists in the prevalence of congenital malformations in IDMs. Recent animal studies have shown that these malformations are related to maternal hyperglycemia early in the pregnancy, and that if it is controlled with insulin during the critical period of embryologic development, then the malformations may be prevented.

Insulin receptors and insulin modulation of norepinephrine uptake in neuronal cultures from rat brain.

Neuronal cells from 1-day-old rat brain in primary culture have been utilized in the present study to characterize insulin-binding sites and a possible action of insulin on these cells. Binding of 125I-insulin to neuronal cultures was 90% specific and time-dependent and reached equilibrium in 120 min. Specific binding was reversible with greater than 90% of binding dissociable within 120 min with a t1/2 of dissociation of 15 min. Various insulin analogues competed for 125I-insulin binding to neuronal cultures according to their known biological potencies. Scatchard analysis of competition data yielded a typical curvilinear plot providing a class of high affinity (Kd = 11 nM) and low affinity (Kd = 65 nM) binding sites. Light microscopic autoradiographic analysis of 125I-insulin bound to neuronal cultures revealed the presence of silver grains predominantly on the neurites with occasional occurrence on the cell soma. Insulin had no effect on neuronal 2-deoxyglucose uptake in contrast with our previous findings demonstrating a 2-fold stimulation of 2-dGlc uptake into astrocyte glial cells from rat brain (Clarke, D.W., Boyd, F.T., Jr., Kappy, M.S., and Raizada, M. K. (1984) J. Biol. Chem. 259, 11672-11675). Incubation of neuronal cultures with insulin caused a dose-dependent inhibition of [3H]norepinephrine uptake with significant inhibition occurring at 1.67 X 10(-11) M. These findings demonstrate that: 1) neuronal cells in primary culture possess specific insulin receptors which are predominantly located on neurites and 2) insulin modulates monoamine uptake in these cultures which suggests that insulin may modulate neural signaling via specific neuronal insulin receptors.

Developing rat brain binds monoiodinated insulin isomers similarly to other extrahepatic target tissues.

We recently reported a series of binding and metabolic studies which led to the conclusion that the developing rat brain is a target tissue for insulin. Since insulin target tissues (extrahepatic) are capable of differentiating between various monoiodoinsulin isomers, we measured the binding of the B26 monoiodoinsulin isomer compared to the A14 in newborn rat brain preparations to determine if the developing rat brain shared the same relative binding of these isomers (viz. B26 greater than A14) with other extrahepatic tissues. The B26 isomer bound 1.57, 1.50 and 1.34 times as much as did the A14 to brain membranes, glia and neurons, respectively, whereas both isomers were bound equally by liver plasma membranes. Competition-inhibition curves were generated using homologous unlabeled (127I) insulin isomers. Binding of the B26 isomer was greater than the A14 at all concentrations. Scatchard plots showed that the receptor concentrations for the two isomers were similar, and affinity profiles showed that the differences in binding could be accounted for by the greater affinity of the receptors for the B26 isomer. The results indicate that the developing rat brain shares with other extrahepatic insulin target tissues a greater affinity for B26 monoiodoinsulin isomer compared to A14. Future studies of insulin binding should avoid using mixtures of iodinated insulins so that a uniform interpretation of data is made possible.

Insulin stimulates macromolecular synthesis in cultured glial cells from rat brain.

The effect of insulin on macromolecular synthesis in glial cells cultured from brains of 1-day-old rats was studied to investigate the role of insulin in brain growth. Insulin caused a dose-dependent stimulation of protein synthesis (measured by [3H]valine incorporation into protein) that became significant by 7 nM insulin. Maximal stimulation of protein synthesis of 145% of control occurred with 18 nM insulin. Long-term protein synthesis was also stimulated to 136% of control by insulin in a dose-dependent manner after 6 days of insulin incubation. Insulin also stimulated net RNA and DNA synthesis (measured by [3H]uridine and [3H]thymidine incorporation into RNA or DNA, respectively) with significant stimulation by 2 nM insulin. Net RNA synthesis stimulation was maximal at 120% of control by 18 nM insulin. Plateau stimulation of DNA synthesis of 175% of control was reached by 200 nM insulin. The effects of insulin on glial protein and RNA synthesis appear to be mediated completely by the insulin receptor. Insulin, in physiological concentrations, stimulated glial DNA synthesis via its interaction with the insulin receptor (46% of total response). At supraphysiological concentrations insulin may have stimulated DNA synthesis via its cross-reactivity with the insulinlike growth factor I receptor (54% of total response). Thus insulin, at concentrations known to exist in the brain, stimulates the processes necessary for growth in the glial cell and is an important growth factor in the developing rat brain.

Insulin binds to specific receptors and stimulates 2-deoxy-D-glucose uptake in cultured glial cells from rat brain.

The kinetics of 125I-insulin binding and physiological activity of insulin on glial cells cultured from brains of 1-day-old rats have been studied. Binding of 125I-insulin to cultured glial cells was specific, reversible, and time-dependent. Porcine and chicken insulin competed equally for 125I-insulin binding while other hormones or insulin analogs competed in proportion to their insulin-like biological activity. Incubation of glial cultures with insulin resulted in a time- and dose-dependent stimulation of 2-deoxy-D-glucose uptake. Maximal stimulation (190% of control) was observed with 18 nM insulin, and 0.1 nM insulin caused half-maximum effect. The stimulatory effect of insulin on 2-deoxy-D-glucose uptake was due to its effect on Vmax without affecting the Km. These observations suggest that insulin stimulates glucose uptake in glial cells cultured from rat brain, the effect mediated by insulin specific receptors.

Effects of insulin and tunicamycin on neuronal insulin receptors in culture.

We have examined the effect of insulin and tunicamycin, which cause decreases in cell surface insulin receptor numbers in peripheral tissues, on insulin receptors in neuron-enriched brain cell cultures. Incubation with 0.016-0.83 microM insulin for 24 h failed to decrease the specific binding of 125I-insulin in neuron-enriched cultures prepared from whole brains of 1-day-old rats. In contrast, these concentrations of insulin produced a dose-dependent decrease in the binding of 125I-insulin in fibroblastic cultures. Tunicamycin, an antibiotic which inhibits glycosylation of proteins and greatly reduced insulin binding via a reduction in apparent receptor numbers in fibroblastic cultures, caused no decrease in the binding of 125I-insulin in neuron-enriched cultures. These results indicate that brain insulin receptors are not down regulated by insulin and that glycosylation of insulin receptors may not be an important step in the regulation of their surface expression in the brain.