A one-step method for genetic transformation of non-piliated Neisseria meningitidis.

A one-step method to chemically transform non-piliated Neisseria meningitidis strains previously resistant to conventional electrochemical transformation procedures has been developed. This method has been used to generate genetically engineered meningococcal strains disrupted in the structural rmpM gene encoding Rmp.

Meningococcal PorA/C1, a channel that combines high conductance and high selectivity.

Class 1 porins (PorA/C1) from Neisseria meningitidis achieve both high selectivity and high conductance. The channel is highly selective (24:1 Na+ over Cl-), suggesting a highly negatively charged selectivity filter. The trimeric nature of PorA/C1 accounts for part of the enormous conductance in 200 mM NaCl (0.97nS). However, the currents that can be achieved exceed the simple infinite-sink calculation for a pore 0.7 nm in radius (estimated from nonelectrolyte permeability). The conductance is linear with salt activity from 20 mM to 2.0 M NaCl with no sign of saturation at low salt. Impermeant polymers reduce the conductance in a manner consistent with their ability to reduce bulk conductivity. Extrapolating from the known structure of homologous porins, the selectivity filter is likely to be small and localized. If small and highly negatively charged ( approximately 9 charges), the predicted conductance would be an order of magnitude higher than that observed. The rate at which ions reach the selectivity filter seems to limit overall ionic flux. PorA/C1 rectifies strongly, and this rectification can be accounted for by calculated differences in the voltage and concentration profiles in the access regions. Thus, it appears that the conductance of this channel is determined by the access resistance and the selectivity by a highly-conductive filter.

Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein.

A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared from CPS serotypes 6B, 14, 19F, and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild type counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 micrograms CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30,000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular, both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysin-induced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines.

Preclinical studies on a recombinant group B meningococcal porin as a carrier for a novel Haemophilus influenzae type b conjugate vaccine.

In anticipation of future combination vaccines, a recombinant class 3 porin (rPorB) of group B meningococci was evaluated as an alternative carrier protein for a Haemophilus influenzae type b (Hib) polyribosylribotol phosphate (PRP) conjugate vaccine. The use of rPorB may avoid undesirable immunologic interactions among vaccine components, including epitopic suppression from conventional carriers (e.g. tetanus toxoid [TT]), as well as provide desirable immunomodulatory effects. Rats were found to be more reliable and consistent than mice or guinea pigs for studying antibody responses to the Hib conjugates. Different Hib conjugates, Hib-TT and Hib-rPorB, consisting of PRP conjugated by reductive amination to TT or rPorB, were compared in rats. Commercially available, licensed vaccines, HbOC (HibTITER) and PRP-T (OmniHib), were used as reference controls. Maximum geometric mean ELISA IgG titers were obtained in rats after only two doses, showing booster effects for all. However, Hib-rPorB immunization consistently resulted in responses that were 1-2 orders of magnitude greater than those for the other conjugates, including the licensed control vaccines. A maximum 4600-fold rise was observed for Hib-rPorB after two doses, and, unlike the other conjugates, a 100% response rate was always achieved without adjuvant. These results warrant further investigation of Hib-rPorB in combination with DTaP.

Characterization of the structure, function, and conformational stability of PorB class 3 protein from Neisseria meningitidis. A porin with unusual physicochemical properties.

PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710-10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic beta-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.

Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1-1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages (Vo=15 mV) than the other (Vo=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less (n=1.3) than that observed for the other process or for the wild-type channel (n=1.5-1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiological function.

Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity.

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of approximately 1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (Tm = 88 degrees C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60 degrees C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.

C-reactive proteins, limunectin, lipopolysaccharide-binding protein, and coagulin. Molecules with lectin and agglutinin activities from Limulus polyphemus.

In 1964, Levin and Bang discovered that gram-negative bacterial endotoxin could rapidly induce gelation of Limulus amebocyte lysate. This observation has led to the development of the most sensitive and specific method for the detection of bacterial endotoxin in pharmaceuticals and drugs intended for human use. Over 10 years ago, Bang injected endotoxin into young horseshoe crabs and observed a time and dose-dependent coagulation of the whole hemolymph. Limunectin, LEBP-PI, and Limulus CRP are found together with coagulin as part of the hemolymph clot at the time of endotoxin-induced exocytosis of amebocytes. In this manner, these molecules with agglutinin/lectin activities could work in concert to assist in the recognition and eventual removal of invading microorganisms from the circulating system. Although the mechanism of endotoxin-induced clot formation is to a large extent understood, the mechanism of clot dissolution and removal in the Limulus hemolymph remains to be clarified.

Agglutination activity of Limulus polyphemus coagulogen following limited proteolysis.

1. A 14 kDa protein with cell agglutination properties has been purified from endotoxin-activated L. polyphemus amebocyte lysate. Amino terminal sequence analysis indicates that this protein corresponds to a proteolytically cleaved product (coagulin) of coagulogen. 2. Similar cell agglutination activity can be generated, in vitro, by proteolytic cleavage of the coagulogen with either trypsin, endogenous protease or an alpha 2M/enzyme complex isolated from amebocytes. 3. Studies with [125I]-labeled coagulogen showed that only coagulin, not the intact coagulogen, binds to rabbit erythrocytes and formalin-fixed amebocytes. 4. The cell agglutination activity of coagulin towards erythrocytes was not inhibited by various sugars tested, and was not Ca(2+)-dependent. 5. These findings suggest that coagulogen and coagulin are reminiscent of their mammalian counterparts, fibrinogen and fibrin, in their clotting and relative adhesive properties.

Isolation, cDNA cloning, and characterization of an 18-kDa hemagglutinin and amebocyte aggregation factor from Limulus polyphemus.

An 18-kDa hemagglutinin which possesses the property of inducing both aggregation of amebocytes and agglutination of erythrocytes has been isolated from Limulus polyphemus amebocytes and purified by ion exchange chromatography. This nonglycosylated, single chain polypeptide with an M(r) of 18,506 and isoelectric point of 8.3 is stored exclusively in the large secretory granules of amebocytes. Based on the partial N-terminal amino acid sequence of 63 residues, DNA probes have been synthesized for screening a pBR322 cDNA library constructed from Limulus amebocytes. The cDNA coding for this protein reveals the presence of a 19-residue signal peptide preceding the 153-residue open reading frame. Northern blot analysis indicates the presence of a single mRNA species. The primary structure derived from the corresponding cDNA sequence reveals an internal homology consisting of two consensus sequences, Val-Asn-Asp/Ser-Trp-Asp and Glu-Asp-Arg-Arg-Trp. The formation of 5 disulfide bonds between 10 half-cysteines divides the molecule into three looped domains each containing the Glu-Asp-Arg-Arg-Trp repeating unit. One of the novel features of this protein is that it shares 37% identity with a 22-kDa mammalian extracellular matrix protein isolated from fetal bovine skin (Neame, P.J., Choi, H.U., and Rosenberg, L.C. (1989) J. Biol. Chem. 264, 5474-5479). The two proteins exhibit a similar pattern of looped domains, each domain containing a homologous consensus sequence (i.e. Glu-Asp-Arg-Arg-Trp). The overall structure of both proteins seems to be highly related, with the exception of an N-terminal tyrosine-rich region present only in the mammalian extracellular matrix protein. The functional properties of the two proteins are similar in that the Limulus 18-kDa protein agglutinates horse erythrocytes and aggregates Limulus amebocytes, and the mammalian 22-kDa protein is an effective adhesion promoter for dermal fibroblasts. On the basis of these unique properties, the newly characterized hemagglutinin has been termed Limulus 18K agglutination-aggregation factor (18K-LAF).

Androgenic expression in the submandibular gland of zinc-deficient mice.

The effects of zinc deficiency were studied in mice submandibular salivary glands (SMG). Zn-restricted mice (Zn-) were maintained from weaning until adult age (60 days) with a powdered diet containing 3 mg Zn2+/kg. Pair-fed animals (30 mg Zn2+/kg powdered diet) and control animals fed a regular pelleted diet were also used. Total protein content and proteolytic activity of SMG did not differ among the groups, but morphometric evaluations revealed significant alterations in the nucleus/cytoplasm size ratios, most likely due to an absolute reduction in nuclear volume (control = 122.5 +/- 6.4; Zn- = 91.6 +/- 10.5; pair-fed = 125.1 +/- 6.8 microns 3) paralleled by an increase of the height of the duct epithelium (control = 70.5 +/- 3.0; Zn- = 90.5 +/- 4.2; pair-fed = 81.7 +/- 3.0 microns). The altered food consistency could be responsible for these morphological changes. In order to assess the subcellular distribution of SMG androgen receptors in conditions of chronic Zn deficiency, Zn- animals were mated and the F1 generation was fed as their dams until the age of 45 days. Cytosolic (in 105,000 g supernatants) and nuclear (KCl-extracted) SMG receptors were determined with [3H]R1881. The Zn- animals had reduced nuclear/cytosolic ratios of androgen receptors (control = 0.62; Zn- = 0.14), as an indication that chronically deficient Zn intake determines a sort of destabilization of the interactions of androgen-receptor complexes with target cell nucleus.

Purification and characterization of an endotoxin-binding protein with protease inhibitory activity from Limulus amebocytes.

Using a lipopolysaccharide affinity column and ion exchange chromatography, a 12-kDa protein has been purified from Limulus amebocytes. In solid phase binding assays, the radiolabeled protein binds specifically to lipopolysaccharide (LPS) with a Kd value on the order of 10(-7) M. A cDNA coding for this protein has been isolated and sequenced. The amino acid sequence deduced from the cDNA indicates that this protein shares no sequence homology with LPS-binding proteins isolated from different species of vertebrates (Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., and Ulevitch, R. J. (1990) Science 249, 1429-1431) and invertebrates (Aketagawa, J., Miyata, T., Ohtsubo, S., Nakamura, T., Morita, T., Hayashida, H., Miyata, T., Iwanaga, S., Takao, T., and Shimonishi, Y. (1986) J. Biol. Chem. 261, 7357-7365). The binding to LPS can be displaced by the unlabeled 12-kDa protein, polymyxin B, lipid A, and to a lesser extent by D-glucosamine. In whole cell binding assays, the 12-kDa protein has also been shown to bind to Escherichia coli. Using both [14C]casein and a synthetic substrate, the protein has been shown to inhibit the proteolytic activity of trypsin, with an IC50 of approximately 10(-7) M. In the presence of LPS, the antitryptic acitivity of the Limulus endotoxin-binding protein-protease inhibitor remains unaffected. The protein is a major component of the cytoplasmic proteins (1%). Immunocytochemical analysis reveals that this protein exists in the secretory granules of the amebocytes where enzymes and substrates for the clotting cascade reside. Based on the unusual dual functional properties, the newly isolated protein was named a "Limulus endotoxin-binding protein-protease inhibitor" (LEBP-PI).

Limunectin. A phosphocholine-binding protein from Limulus amebocytes with adhesion-promoting properties.

A protein that binds to and precipitates with pneumococcal C-polysaccharide and a phosphocholine (PC) derivative of bovine serum albumin has been affinity purified from Limulus amebocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals that the isolated protein consists of a single polypeptide chain of approximately 50 kDa. It is an intracellular protein localized in the secretory granules of amebocytes according to immunogold staining. Although it shares the PC-binding property with C-reactive protein isolated from Limulus and other animal species, it differs from C-reactive protein in that the latter binds to PC only in the presence of Ca2+, whereas the newly isolated protein binds to PC in a Ca(2+)-independent manner. In this respect, the newly isolated PC-binding protein resembles the antibodies to PC of mouse myelomas. The gene coding for this protein has been isolated. The gene sequence predicts a protein of 54 kDa with an unusual structural feature: it consists almost entirely of 10 contiguous segments, 45 amino acids in length, with extensive homology. Some limited sequence homologies were found between the 54-kDa protein and segments of vitronectin, gelatinase, and collagenase. It binds to bacterial cells, fixed amebocytes, and a number of extracellular matrix molecules. Due to its structural and some functional similarities to other adhesion molecules, the Limulus 54-kDa protein was named "Limunectin."

Effects of zinc on the trophic activity of testosterone in androgen target tissues of castrate mice.

Swiss, 60-day castrate mice were injected with 0.5, 1.5 or 5.0 mg of testosterone propionate (TP; single dose, subcutaneously) 5 days before sacrifice, in order to investigate the ability of the submandibular gland (SMG) and other androgen target tissues to recover their normal morphology and function. Some animals were additionally injected intraperitoneally with ZnCl2 (0.14 or 0.28 mg Zn2+/animal per day) during the last 15 days before sacrifice. Only SMG tissue fully recovered by TP treatment. ZnCl2 significantly impaired the dose-dependent recovery of the granular ducts of mouse SMG tissue and that of other organs which display 'androgenic' (prostate, epididymis) and 'anabolic' responses (bulbocavernosus muscle). Histological examination of testes and epididymides of intact mice injected with ZnCl2 revealed abnormal spermatogenesis with multinucleated cells and acidophilic bodies within the tubular lumen; the circulating levels of testosterone in these animals were low. In vitro, Zn2+ inhibited androgen-binding activity in SMG cytosol, but the binding capacity increased in SMG of zinc-injected animals. It is suggested that zinc, although essential for the androgenic expression, is critical as far as its intracellular concentrations are concerned and that pharmacological doses of Zn2+ determine androgenic suppression by competition at receptor and acceptor levels.

Chronic administration of aqueous extract of Stevia rebaudiana (Bert.) Bertoni in rats: endocrine effects.

1. The effects of the active principles of S. rebaudiana (SR) on endocrine parameters of male rats were studied upon chronic administrations (60 days) of a concentrated, crude extract of its leaves, starting at prepubertal age (25-30 days old). 2. The following determinations were made: glycemia; serum levels of T3 and T4; available binding sites in thyroid hormone-binding proteins (T3R index); binding of [3H]R 1881 to prostate cytosol; zinc content in prostate, testis, submandibular salivary gland (SMG) and pancreas; water content in testis and prostate. The body weight gain and the final weight of testis, prostate, seminal vesicle, SMG and adrenal were also studied. 3. Results showed that the SR-treated group did not significantly differ from the control group, with exception to the seminal vesicle weight, which fell by about 60%. 4. It is concluded that if the SR extract does have some potential to decrease rat fertility at all, this effect is almost certainly not exerted on the male.

Thyroidal modulation of androgenic expression in mice submandibular gland.

We investigated the influence of testes and thyroid gland on the maintenance of biochemical parameters and of [3H]R1881 binding sites of adult mice submandibular gland (SMG). Castration (Cx) performed at beginning of puberty prevented sex-dependent SMG development without interfering with maximal androgen binding capacity. Thyroidectomy (Tx) had strong effects on SMG, mainly by lowering the number of androgen binding sites. All alterations could be fully reverted after treatment with testosterone (5 mg/animal, single dose) or with thyroxine (T4, 250 micrograms/animal per day during 5 days). The effects of Cx on SMG could be reverted by therapy with testosterone, T4, or with both hormones (testosterone + T4) in a non-synergistic fashion. It is shown the importance of thyroidal activity on the physiological maintenance of androgen receptors in the murine SMG; the role played by thyroid gland seems to be essential for the full expression of the androgen-dependent SMG activity in adult mice.

Ontogenesis of androgen receptors in the mouse submandibular gland: correlation with the developmental profiles of circulating thyroid and testicular hormones.

Specific binding of the synthetic androgen, [17 alpha-methyl-3H]methyltrienolone, to the cytosol fraction of the submandibular salivary gland (SMG) of male mice was studied in relation to the developmental profiles of testosterone and thyroid hormones in blood. The peak rise of serum triiodothyronine (T3) at prepubertal age was closely related to both the increase of maximal androgen-binding capacity in SMG and the conspicuous surge of proliferative activity as indicated by increased rate of glandular DNA content. Also, 2-month thyroidectomized mice had an age-related, strong reduction in the number of androgen-binding sites. On the other hand, the development of the secretory functions of the gland could be better related to the rise of circulating testosterone by days 25-30 of age. The results suggest that thyroid hormones play a very important role in the early induction and further maintenance of androgen receptors in the murine SMG.

Differential actions of testosterone and its metabolites on mice submandibular gland.

Submandibular glands from male mice castrated at 21 days of age and killed 60 days thereafter exhibited impaired development of the granular ducts (GD), an effect which is directly related to its androgen-dependent feature. Testosterone and other related steroids of natural occurrence were given to these animals in order to determine the mechanism by which those compounds bring about recovery of their glandular histophysiology. The size and number of GD were reconstituted by the steroids in the following order of potency: 5 alpha-androstane-3 alpha, 17 beta-diol greater than testosterone propionate greater than 5 alpha-dihydrotestosterone greater than or equal to testosterone greater than 5 alpha-androstane-3 beta, 17 beta-diol. All of the drugs stimulated protein synthesis, but only 3 alpha-diol was also able to increase DNA synthesis. Results support the assumption that 3 alpha-diol exerts both proliferative and hypertrophic effects on mice SMG, possibly through some receptor-independent pathway(s); the action of the other steroids is essentially hypertrophic in nature.

Beta-adrenergic stimulation of mouse parotid gland: amylase activity and secretory granules during isoproterenol-induced mitosis.

This paper examines the mitotic activity of parotid glands of groups of mice sacrificed at 1 hr-intervals between 26 and 36 hr after being injected with dl-isoproterenol (IPR, 1 mumol/g, single dose, i.p.). Maximal mitotic activity occurs 35 hr after IPR injection; there is a concomitant rise of amylase activity (64% over control). Mitotic cells showed increased number of secretory granules (N) with regard to interphasic cells. However, the increment of N is paralleled by an increment of the cell section area (A); the N/A ratio is thus maintained around 0.45 throughout. These findings suggest that during IPR-induced mitosis of mice parotid cells there is not a blockade of the biosynthetic pathways which lead to the appearance of secretory granules in the cell cytoplasm.

Effects of testosterone and its metabolites in relation to androgen-binding activity in murine submandibular salivary glands.

The relative potencies of testosterone (T), testosterone propionate (TP) and other related steroids (5 alpha-dihydrotestosterone, DHT; 5 alpha-androstane-3 alpha, 17 beta-diol, alpha-diol; 5 alpha-androstane-3 beta, 17 beta-diol, beta-diol) in restoring some morphological and functional characteristics of submandibular gland (SMG) were investigated in castrated mice. The steroids restored to near-control values the glandular weight and total protein content, alpha-diol and TP being the most effective compounds tested; with regard to proteolytic activity stimulation, beta-diol was more effective than DHT. The alpha-diol metabolite was unique in significantly increasing DNA synthesis. In competition studies, alpha-diol and beta-diol were ineffective in displacing the specifically bound [3H]-R1881 from the SMG androgen-binding macromolecules. Thus T can elicit its effects on SMG without the need of prior conversion to DHT; there must be alternative, receptor-independent mechanisms whereby alpha- and beta-diol exert trophic/metabolic effects on murine SMG.