Mannose Molecules Research Articles

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Mannose-Specific Lectin CGL1 from the Pacific Oyster Crassostrea gigas.

A novel mannose-specific lectin, named CGL1 (15.5 kDa), was isolated from the oyster Crassostrea gigas. Characterization of CGL1 involved isothermal titration calorimetry (ITC), glycoconjugate microarray, and frontal affinity chromatography (FAC). This analysis revealed that CGL1 has strict specificity for the mannose monomer and for high mannose-type N-glycans (HMTGs). Primary structure of CGL1 did not show any homology with known lectins but did show homology with proteins of the natterin family. Crystal structure of the CGL1 revealed a unique homodimer in which each protomer was composed of 2 domains related by a pseudo two-fold axis. Complex structures of CGL1 with mannose molecules showed that residues have 8 hydrogen bond interactions with O1, O2, O3, O4, and O5 hydroxyl groups of mannose. The complex interactions that are not observed with other mannose-binding lectins revealed the structural basis for the strict specificity for mannose. These characteristics of CGL1 may be helpful as a research tool and for clinical applications.

CdTe quantum dots conjugated to concanavalin A as potential fluorescent molecular probes for saccharides detection in Candida albicans

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

Quantitative assessment of the multivalent protein-carbohydrate interactions on silicon.

A key challenge in the development of glycan arrays is that the sensing interface be fabricated reliably so as to ensure the sensitive and accurate analysis of the protein-carbohydrate interaction of interest, reproducibly. These goals are complicated in the case of glycan arrays as surface sugar density can influence dramatically the strength and mode of interaction of the sugar ligand at any interface with lectin partners. In this Article, we describe the preparation of carboxydecyl-terminated crystalline silicon (111) surfaces onto which are grafted either mannosyl moieties or a mixture of mannose and spacer alcohol molecules to provide "diluted" surfaces. The fabrication of the silicon surfaces was achieved efficiently through a strategy implicating a "click" coupling step. The interactions of these newly fabricated glycan interfaces with the lectin, Lens culinaris, have been characterized using quantitative infrared (IR) spectroscopy in the attenuated total geometry (ATR). The density of mannose probes and lectin targets was precisely determined for the first time by the aid of special IR calibration experiments, thus allowing for the interpretation of the distribution of mannose and its multivalent binding with lectins. These experimental findings were accounted for by numerical simulations of lectin adsorption.

(Oligo)mannose functionalized hydroxyethyl starch nanocapsules: en route to drug delivery systems with targeting properties.

Hydroxyethyl starch nanocapsules (NCs) are potentially interesting hydrophilic drug delivery carriers, since they do not show non-specific interactions with the living cells. Only the presence of a targeting agent on their surface allows them to target specifically the desired site of action. In this paper, we report the synthesis and cell uptake of crosslinked hydroxyethyl starch (HES) NCs decorated with (oligo)mannose, which is an effective targeting agent for macrophage and dendritic cells. The crosslinked HES NCs were prepared via the interfacial polyaddition of HES with 2,4-toluene diisocyanate (TDI) in inverse (water-in-oil) miniemulsion and then functionalized with (oligo)mannose following two different strategies. To compare the activity and availability of a targeting agent, different types of mannose molecules such as α-d-mannopyranosylphenyl isothiocyanate, 3-O-(α-d-mannopyranosyl)-d-mannose and α3,α6-mannotriose were used for the functionalization of NCs. The availability of the mannose was unambiguously assessed by interaction with a fluorescent lectin. Moreover, the accessibility of the pilot molecule was improved by the presence of a PEG linker at the surface of the NCs. To simulate in vivo conditions, where proteins interact with nanoparticles with a possible hindrance of the accessibility to the targeting agent, the mannosylated NCs were first incubated with human serum before interaction with the fluorescent lectin. Enhancement of uptake into dendritic cells demonstrates the targeting ability in in vitro studies.

Optimization of Localized Surface Plasmon Resonance Transducers for Studying Carbohydrate–Protein Interactions

Noble metal nanostructures supporting localized surface plasmons (SPs) have been widely applied to chemical and biological sensing. Changes in the refractive index near the nanostructures affect the SP extinction band, making localized surface plasmon resonance (LSPR) spectroscopy a convenient tool for studying biological interactions. Carbohydrate-protein interactions are of major importance in living organisms; their study is crucial for understanding of basic biological processes and for the construction of biosensors for diagnostics and drug development. Here LSPR transducers based on gold island films prepared by evaporation on glass and annealing were optimized for monitoring the specific interaction between Concanavalin A (Con A) and D-(+)-mannose. The sugar was modified with a PEG-thiol linker and immobilized on the Au islands. Sensing assays were performed under stationary and flow conditions, the latter providing kinetic parameters for protein binding and dissociation. Ellipsometry and Fourier transform-infrared (FT-IR) data, as well as scanning electron microscopy (SEM) imaging of fixated and stained samples, furnished independent evidence for the protein-sugar recognition. Enhanced response and visual detection of protein binding was demonstrated using Au nanoparticles stabilized with the linker-modified mannose molecules. Mannose-coated transducers display an excellent selectivity toward Con A in the presence of a large excess of bovine serum albumin (BSA).

English

Tuberculosis is the second lethal infectious disease caused by Mycobacterium species. This pathogens could cause severe disease like tuberculosis and leprosy in human. Today there are a number of anti tuberculosis agents utilized in treatment of this disease but multiple drug resistance is one of the major problems that end to failure in treatment. Dendrimers are synthetic, high branched polymers with a number of functional groups that could bind to different macromolecules like drugs, oligosaccharides that makes it appropriate for target drug delivery. Today, some type of dendrimers like Jeffamines are developed possessing self antimicrobial activity. Hypothesis: we propose that combination of ethambutol with 3rd generation of Jeffamine core based dendrimer (P3) and mannose could both create a complex compound with high potency against mycobacteriumspecies that is targeted to macrophages via interaction between lectin receptors on immune cells and mannose molecules on outer branch of P3.   Key words: Tuberculosis, dendrimers, Jeffamines, Mycobacterium, ethambutol.

Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45–13.3 PEG chains and 4.7–3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.

Electrochemical cytosensor based on gold nanoparticles for the determination of carbohydrate on cell surface

An electrochemical cytosensor was designed based on the specific recognition of mannosyl on a cell surface to concanavalin A (ConA) and the signal amplification of gold nanoparticles (NPs). By sandwiching a cancer cell between a gold electrode modified with ConA and the gold NPs with ConA and 6-ferrocenylhexanethiol (Fc), the electrochemical cytosensor was established. The cell number and the amount of cell surface mannose moieties were quantified by cyclic voltammetry (CV) analysis of the Fc loaded on the surface of the gold NPs. Since a single gold NP could be loaded with hundreds of Fc, a significant amplification for the detection of target cell was obtained. By using K562 leukemic cells (K562 cells) as a model, the electrochemical response was proportional to the cell concentration in the range from 1.0 × 10 2 to 1.0 × 10 7 cells mL −1, showing very high sensitivity. The signal amplification could be further used to evaluate the cell surface mannose moieties, and the amount of mannose moieties on a single living K562 cell was detected to correspond to 4.7 × 10 9 molecules of free mannose. This strategy presents a promising platform in a highly sensitive cytosensor and convenient estimation of cell surface carbohydrate.

Mono‐, Di‐, or Triazidated Cyclodextrin‐Based Polyrotaxanes for Facile and Efficient Functionalization via Click Chemistry

As a feasible way for controlling the density of ligands in polyrotaxanes, azidated polyrotaxanes comprising PEG (MW= 3,000 and 20,000 g · mol(-1)) and mono-, di-, or triazidated α-cyclodextrins are prepared in a water/DMSO solution in a one-pot synthesis. The azidated polyrotaxanes are then allowed to conjugate with propargyl-modified mannose as a ligand via click chemistry. As proven by FTIR spectroscopy and (1)H NMR-spectroscopy, mannose molecules are efficiently introduced into all of the azide moieties of the polyrotaxanes. The results verify the achievement of ligand-density-controlled polyrotaxanes. The functionalized polyrotaxanes can be utilized for a variety of biological applications.

Effective Cell Capture with Tetrapeptide-Functionalized Carbon Nanotubes and Dual Signal Amplification for Cytosensing and Evaluation of Cell Surface Carbohydrate

A novel electrochemical cytosensing strategy was designed based on the specific recognition of integrin receptors on a cell surface to arginine-glycine-aspartic acid-serine (RGDS)-functionalized single-walled carbon nanotubes (SWNTs). The covalent conjugation of the RGDS tetrapeptide to SWNTs was proved with Raman and FT-IR spectra. The conjugated RGDS showed a predominant ability to capture cells on the electrode surface by the specific combination of RGD domains with integrin receptors. With the use of BGC-823 human gastric carcinoma cells (BGC cells) as a model, the cell surface mannosyl groups could specifically bind with horseradish peroxidase labeled concanavalin A, producing an electrochemical cytosensor. On the basis of the dual signal amplification of SWNTs and enzymatic catalysis, the cytosensor could respond down to 620 cells mL (-1) of BGC cells with a linear calibration range from 1.0 x 10 (3) to 1.0 x 10 (7) cells mL (-1), showing very high sensitivity. The dual signal amplification could be further used to evaluate the mannosyl groups on the cell surface, and the mannosyl groups on a single living intact BGC cell were detected to correspond to 5.3 x 10 (7) molecules of mannose. This strategy presents a promising platform for highly sensitive cytosensing and convenient evaluation of surface carbohydrates on living cells.

Saccharomyces cerevisiaeα1,6‐mannosyltransferase has a catalytic potential to transfer a second mannose molecule

In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.

Crystal structure of a carbohydrate induced homodimer of phospholipase A 2 from Bungarus caeruleus at 2.1 Å resolution

This is the first crystal structure of a carbohydrate induced dimer of phospholipase A 2 (PLA 2). This is an endogenous complex formed between two PLA 2 molecules and two mannoses. It was isolated from Krait venom ( Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA 2 was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0–2.1 Å. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA 2 molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA 2 molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.

Development of 99mTc-neomannosyl human serum albumin (99mTc-MSA) as a novel receptor binding agent for sentinel lymph node imaging

Various mannose receptor-binding agents, for example 99mTc-diethylenetriaminepentaacetic acid (DTPA)-mannosyl-polymer, have been developed for sentinel lymph node (SLN) imaging. In order to simplify the synthesis and labelling procedure and to improve the biological properties, we developed a novel mannose receptor-binding agent, 99mTc-neomannosyl human serum albumin (99mTc-MSA), for SLN imaging. MSA was synthesized by conjugating mannopyranosylphenylisothiocyanate to human serum albumin (HSA). After reducing MSA with beta-mercaptoethanol and PD-10 column purification, a medronate solution containing stannous fluoride was added, divided into aliquots and freeze-dried. Reduced MSA was labelled with 99mTc-pertechnetate solution. The stability was checked for 24 h at 37 degrees C in human serum. The biodistribution of 99mTc-MSA in mice was investigated by intravenous injection through the tail vein and subcutaneous injection into the foot pad. The biodistributions of 99mTc-HSA and 99mTc-antimony sulphur colloid (99mTc-ASC) were also investigated for comparison. Dynamic whole-body images were obtained for 30 min after subcutaneous injection into the rats' foot pads. The number of mannose molecules conjugated per MSA was 15.9. The number of thiol groups produced was 19.4 per MSA after reduction with beta-mercaptoethanol. Labelling yields were always higher than 97%. 99mTc-MSA was stable for 24 h at 37 degrees C in human serum. The biodistribution in mice after intravenous injection showed high liver uptake (50.7+/-5.5% and 42.7+/-3.7% injected dose per gram at 10 and 60 min, respectively). 99mTc-MSA and 99mTc-ASC showed high accumulation in the lymph nodes after subcutaneous injection, whereas 99mTc-HSA and Tc-tin colloid did not, in both biodistribution and imaging studies. We have successfully developed a novel 99mTc-MSA for lymphoscintigraphy. The results of animal studies show that 99mTc-MSA has promising properties for SLN imaging.

Evidence That Intracellular β1-2 Mannan Is a Virulence Factor in Leishmania Parasites

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.

Crystal Structure of a Dimeric Mannose-specific Agglutinin from Garlic: Quaternary Association and Carbohydrate Specificity

A mannose-specific agglutinin, isolated from garlic bulbs, has been crystallized in the presence of a large excess of α- d -mannose, in space group C 2 and cell dimensions, a=203.24, b =43.78, c=79.27 Å, β=112.4 °, with two dimers in the asymmetric unit. X-ray diffraction data were collected up to a nominal resolution of 2.4 Å and the structure was solved by molecular replacement. The structure, refined to an R -factor of 22.6 % and an R freeof 27.8 % reveals a β-prism II fold, similar to that in the snowdrop lectin, comprising three antiparallel four-stranded β-sheets arranged as a 12-stranded β-barrel, with an approximate internal 3-fold symmetry. This agglutinin is, however, a dimer unlike snowdrop lectin which exists as a tetramer, despite a high degree of sequence similarity between them. A comparison of the two structures reveals a few substitutions in the garlic lectin which stabilise it into a dimer and prevent tetramer formation. Three mannose molecules have been identified on each subunit. In addition, electron density is observed for another possible mannose molecule per dimer resulting in a total of seven mannose molecules in each dimer. Although the mannose binding sites and the overall structure are similar in the subunits of snowdrop and garlic lectin, their specificities to glycoproteins such as GP120 vary considerably. These differences appear, in part, to be a direct consequence of the differences in oligomerisation, implying that variation in quaternary association may be a mode of achieving oligosaccharide specificity in bulb lectins.

Molecular heterogeneity of the beta-core fragment of human chorionic gonadotrophin.

We have analysed the structure and composition of the beta-core fragment of human chorionic gonadotrophin (beta C-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced beta C-hCG demonstrated two major bands with apparent molecular weights of M(r) 8900 and M(r) 7500. The molecular weight of the agalacto beta C-hCG was estimated to be M(r) 10,218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported beta 6-40 (M(r) 5000) and beta 55-92 (M(r) 5300) peptides of the beta hCG subunit. The results showed that 56-78% of beta C-hCG molecules of molecular weight M(r) 12,800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2.8, glucosamine 5.3, galactosamine 0.3, fucose 1.7 and sialic acid 3.0. Approximately 22-44% of the beta C-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately M(r) 10,000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of beta C-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues.

Liposome targeting to mouse brain: Mannose as a recognition marker

Liposomes prepared from lecithin:cholesterol:p-aminophenyl-alpha-mannoside (7:2:1, v/v/v) were efficiently incorporated into the mouse brain across the blood brain barrier. Furthermore, liposomes injected intraperitoneally were exclusively distributed into lysosome rich fraction and also taken up by glial cells. These data suggest that blood brain barrier cells and glial cells recognize mannose molecule on the surface of the membrane and can be used for the treatment of brain damage by lysosomal storage disease.

Identification of two chemical types of K21 capsular polysaccharide from klebsiellae.

Strains of Klebsiella pneumoniae of serotype K21 are frequently involved in outbreaks of nosocomial infections. The type strain of Klebsiella pneumoniae K21 (which we have renamed K21a) produces capsular polysaccharide that contains mannose, galactose and glucuronic acid in the ratio 2:2:1. In contrast, all eight of the randomly chosen isolates of Klebsiella pneumoniae that were initially typed as K21 were shown by paper chromatography and NMR spectroscopy to produce a different capsular polysaccharide. We have designated this new polysaccharide K21b. The K21b capsular material appears to have a closely similar immunodominant side chain to K21a. However, K21b polysaccharide has two molecules of rhamnose in the polysaccharide backbone replacing the two molecules of mannose found in the K21a capsule. Our results suggest that the K21b Klebsiella serotype may be more frequently distributed than the classical K21a type.

Influence of Boron Nutrition on Cell Wall Polysaccharides in Cell Cultures of Daucus carota L.

Summary Suspension cultured cells of Daucus carota contained high amounts of non-cellulosic cell wall components. Only about 10 % of the total carbohydrates could be attributed to cellulose. Boron deficiency for two and five days resp. reduced the incorporation of 14 C-glucose into cellulosic polysaccharides as well as the total cellulose content in crude cell wall preparations. Under B-deficiency molar ratios between individual sugar components remained rather constant with the exception of the mannose to glucose ratio in the fraction not hydrolyzed by 2M TFA («cellulosic aldoses»). As the mannose molecule exhibits two hydroxyl-groups in cis-position, boric acid may directly or indirectly influence polysaccharide synthesis by forming complexes with mannose. Moreover, B may affect the deposition of cell wall material by altering membrane properties. The changes in cell wall polysaccharide composition induced by B deficiency are relatively small in Daucus cells and are thus not the primary cause for the growth inhibition observed.

Glucose stimulation of testosterone-maintained protein synthesis in the rat ventral prostate

The stimulatory effect of exogenous glucose on protein synthesis maintained by testosterone was studied by measuring the in vitro incorporation of amino acids into proteins in the rat ventral prostate. The addition of glucose did not influence the specific activity of the extracellular or intracellular amino acid pools. The amount of newly-synthetized proteins was increased in the presence of glucose. Mannose was as effective as glucose, whereas a variety of other substrates (e.g. pyruvate, lactate, amino acids, acetate) which are readily metabolized by the prostate, failed to support to the same extent hormoneactivated protein synthesis. None of the substrates increased protein synthesis in the prostate of castrated rats. The incubation of the prostate without any substrate led to a decline in the level of ATP, the ATP/ADP ratio and the adenylate energy charge and to a rise in the levels of ADP and AMP irrespective of the hormonal state of the tissue. The addition of glucose restored the levels and ratios of the adenine nucleotides, but several other substrates were also able to maintain the initial ATP levels in the prostate of castrated, as well as castrated, testosterone-treated rats. The stimulatory effect of glucose on hormone-maintained protein synthesis cannot be explained by changes in the adenine nucleotides. Nevertheless, the activation of glucose metabolism may play an important role by providing energy for increased synthetic reactions. Moreover, glucose and mannose molecules themselves or a glycolytic metabolite (proximal to pyruvate) may be required for the full expression of the transcriptional effect of testosterone on protein synthesis in the ventral prostate of castrated rats.