The use of a collagen matrix in hard-to-heal venous leg ulcers.

OBJECTIVE: The effects of a collagen dressing on hard-to-heal venous leg ulcers (vlUs) were evaluated in this prospective, randomised, controlled study. METHOD: Patients with hard-to-heal vlU were included and divided into two groups using the block randomisation method. The first group was treated with a collagen and an alginate dressing (group A), and the second group with an alginate dressing alone (group b). both groups also had a short-stretch compression system applied at every dressing change. The dressings were changed twice a week for 12 weeks or until the ulcer was healed. Granulation tissue improvement, wound size, overall dressing performance and dressing comfort were evaluated and recorded. RESULTS: A total of 40 patients completed the study evaluation period. Group A had a 65% increase in granulation tissue compared to 38% in group b. The mean ulcer area was reduced to 45% in group A compared to 20% in group b at 12 weeks. no significant side effects were detected in either group. Patients of both groups were satisfied with their treatment and healing progress. CONCLUSION: The results of this study showed the effectiveness and safety of a collagen dressing in hard-to-heal vlUs as an adjunctive therapy with compression bandaging. These encouraging results may positively affect the quality of life of patients with chronic wounds.

Development of an HPLC/UV assay for the evaluation of inhibitors of human recombinant monoacylglycerol lipase.

Monoacylglycerol lipase (MAGL) is a membrane-associated cytosolic serine hydrolase which catalyses the hydrolysis of the endocannabinoid 2-arachidonoylglycerol into arachidonic acid and glycerol. MAGL represents the link between the endocannabinoid and the eicosanoid system indeed its inhibition enhances endocannabinoid signalling and lowers eicosanoid production. Here we present a radioactive-free, sensitive and solid HPLC-UV based method to evaluate MAGL activity by using 4-nitrophenylacetate (4-NPA) as substrate. The enzymatic activity is measured by quantifying the 4-nitrophenol (PNP) (λ = 315 nm) formation on a C18 stationary phase. The method was validated by calculating IC50 values of the reference inhibitors JZL184, CAY10499 and JW642 and confirming the irreversible and non-competitive mechanism of inhibition for JZL184. Furthermore in order to resemble the catalytic conditions of MAGL at cell membrane level, the surfactant Triton X-100 was added, as a micelle forming agent and 4-nitrophenyldodecanoate (4-NPDo) was used as lipophilic substrate for MAGL. The data obtained confirmed that the HPLC method is an alternative, radioactive-free approach for the screening and characterization of new MAGL inhibitors. Finally this assay prevents, in an unequivocal manner, any interference related to the intrinsic absorbance of screened compounds or metabolites generated upon enzymatic cleavage which could seriously affect the assay readout.

Simultaneous detection and quantification of parecoxib and valdecoxib in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology.

Parecoxib is the injectable prodrug of valdecoxib, a cicloxygenase-2 selective drug, currently used in human medicine. Recent studies have suggested both its excellent clinical effectiveness and wide safety profile. The aim of the present study was to develop and validate a new high-performance liquid chromatography (HPLC) with spectrofluorimetric detection method to quantify parecoxib and valdecoxib in canine plasma. Several parameters both in the extraction and the detection method were evaluated. The applicability of the method was determined by administering parecoxib to one dog: the protocol provided the expected pharmacokinetic results. The final mobile phase was acetonitrile: AcONH(4) (10 mM; pH 5.0) 55:45, v/v, with a flow rate of 0.4 mL min(-1), and excitation and emission wavelengths of 265 and 375 nm, respectively. The analytical column was a reverse-phase C18 ODS2 3-µm particle size. Protein precipitation in acidic medium followed by two successive liquid-liquid steps was carried out. The best extraction solvent was cyclohexane:Et(2)O (3:2, v/v) that gave recoveries ranging from 81.1% to 89.1% and from 94.8% to 103.6% for parecoxib and valdecoxib, respectively. The limits of quantification were 25 and 10 ng mL(-1) for parecoxib and valdecoxib, respectively. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC-mass spectrometry experiments. The other validation parameters were in agreement with the European Medicines Evaluation Agency and International Conference on Harmonisation guidelines. In conclusion, this method (extraction, separation and applied techniques) is simple and effective. This is the first time that use of a HPLC with spectrofluorimetric detection technique to simultaneously detect parecoxib and valdecoxib in plasma has been reported. This technique may have applications for pharmacokinetic studies.

[Accuracy of pedicle screw insertion in the thoracolumbar spine using image-guided navigation]. / Fiabilidad del navegador en la colocación de tornillos pediculares toracolumbares.

INTRODUCTION: Computer image guidance is one of the most significant technologic advancements in the spine surgery, because preoperative or intraoperative images can be used for multiplanar, three-dimensional intraoperative navigation. MATERIAL AND METHODS: We performed a prospective clinical study to assess the accuracy of pedicle screw insertion using an optoelectronic navigation system (SurgiGATE Spine 2.1 Medivision). The study population included 29 patients with diverse disorders of the thoraco- lumbar spine (degenerative 54%, spondylolisthesis 21%, fractures 14%, scoliosis 7% and spondylodiscitis 4%). One patient was excluded from the study because problems with the specific instruments or the computer system. Pre and post-operative axial computed tomography images were obtained for each patient and analyzed by two independent radiologists to placement accuracy. The correct location was defined accord to Heary scale in 5 grades. RESULTS: 163 image-guided thoraco-lumbar pedicle screws were placed 29 in the thoracolumbar spine and 134 in the lumbosacral spine. We achieved a completely intraosseous placement (Grade I) in 99.4% of lumbosacral spine screws and 100% of thoracolumbar spine screws. Only one misplaced screw (Grade III) in the pedicle of L III in the concavity of a scoliosis was reported. No implant related complications were noted. CONCLUSIONS: The low rate of misplaced screws in this prospective study compares favorably with previously published results. Our initial results indicate that Image-guided spinal surgery is a safe technique which improves surgical performance during posterior transpedicle stabilization.

Fiabilidad del navegador en la colocación de tornillos pediculares toracolumbares / Accuracy of pedicle screw insertion in the thoracolumbar spine using image-guided navigation

Introducción. La cirugía guiada por imagen es uno de los más importantes avances tecnológicos dentro de la cirugía del Raquis ya que permite al cirujano realizar una navegación multiplanar tridimensional en tiempo real en el interior de una vértebra. Material y métodos. Realizamos un estudio clínico prospectivo no randomizado sobre la fiabilidad en la colocación de tornillos pediculares mediante un sistema de navegación optoelectrónico (SurgiGATE Spine 2.1 Medivision). Se estudiaron veintinueve pacientes intervenidos por diferentes patologías en columna toracolumbar incluyendo: degenerativas (54%), espondilolistesis(21%), fracturas (14%), escoliosis (7%) y espondilodiscitis(4%). Un paciente fue eliminado del estudiodebido a un fallo técnico en el equipo de navegación.Se obtuvieron imágenes de TC pre y postoperatorias de cada paciente y éstas fueron evaluadas por dos neurorradiólogos independientes. La colocación correcta se definió de acuerdo a la escala de Heary en 5 grados.Resultados. Se colocaron 163 tornillos, 29 en la columnatoracolumbar y 134 en la columna lumbosacra.Hemos conseguido una colocación totalmente intraósea(Grado I) en el 99,4% de tornillos en la columnalumbosacra y en un 100% en la columna toracolumbar. Se comprobó el error de colocación (Grado III) en un pedículo de L3 en la concavidad de una escoliosis.No se observaron complicaciones relacionadas con los implantes.Conclusiones. El bajo porcentaje de tornillos mal colocados en este estudio se compara favorablemente con los resultados publicados en la literatura. Nuestros resultados indican que la cirugía guiada por imagen aplicada a la cirugía del raquis es una técnica segura para la fijación transpedicular (AU)

Introduction. Computer image guidance is one of the most significant technologic advancements in the spinesurgery, because preoperative or intraoperative images can be used for multiplanar, three-dimensional intraoperative navigation. Material and methods. We performed a prospective clinical study to assess the accuracy of pedicle screw insertion using an optoelectronic navigation system (SurgiGATE Spine 2.1 Medivision). The study population included 29 patients with diverse disorders of the thoracolumbar spine (degenerative 54%, spondylolisthesis 21%, fractures 14%, scoliosis 7% and spondylodiscitis 4%). One patient was excluded from the study because problems with the specific instruments or the computer system. Pre and post-operative axial computed tomography images were obtained for each patient and analyzed by two independent radiologists to place mentaccuracy. The correct location was defined accord to Heary scale in 5 grades. Results. 163 image-guided thoraco-lumbar pedicle screws were placed 29 in the thoracolumbar spine and 134 in the lumbosacral spine. We achieved a completely intraosseous placement (Grade I) in 99.4% of lumbosacral spine screws and 100% of thoracolumbar spinescrews. Only one misplaced screw (Grade III) in the pedicle of L III in the concavity of a scoliosis was reported. No implant related complications were noted. Conclusions. The low rate of misplaced screws in this prospective study compares favorably with previously published results. Our initial results indicate that Image-guided spinal surgery is a safe technique which improves surgical performance during posterior transpedicle stabilization (AU)

Study of apoptosis induction and deoxycytidine kinase/cytidine deaminase modulation in the synergistic interaction of a novel ceramide analog and gemcitabine in pancreatic cancer cells.

This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.

Determination of tramadol and metabolites by HPLC-FL and HPLC-MS/MS in urine of dogs.

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC-MS/MS. A single oral dose of tramadol (4mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively. The present method allowed to obtain good selectivity, accuracy, precision and recoveries without the need of time consuming or expensive clean up steps. The short chromatographic time courses allowed this analysis to be proposed for routine purposes. The HPLC-MS/MS detected in the urine two metabolites (M6 and M7) considered negligible in humans. The low LOQ showed that the method could be useful for the determination of the illegal use of this drug in race-dogs' urine.

Inhibitors of lactate dehydrogenase isoforms and their therapeutic potentials.

In many different species, lactate dehydrogenase (LDH) constitutes a major checkpoint of anaerobic glycolysis, by catalyzing the reduction of pyruvate into lactate. This enzyme has recently received a great deal of attention since it may constitute a valid therapeutic target for diseases so different as malaria and cancer. In fact, the isoform expressed by Plasmodium falciparum (pfLDH) is a key enzyme for energy generation of malarial parasites. These species mostly depend on anaerobic glycolysis for energy production, since they lack a citric acid cycle for ATP formation. Therefore, inhibitors of pfLDH would potentially cause mortality of P. falciparum and, to this purpose, several small organic molecules have been recently designed and developed with the aim of blocking this new potential antimalarial chemotherapeutic target. Moreover, most invasive tumour phenotypes show a metabolic switch (Warburg effect) from oxidative phosphorylation to an increased anaerobic glycolysis, by promoting an upregulation of the human isoform-5 of lactate dehydrogenase (hLDH-5 or LDH-A), which is normally present in muscles and in the liver. Hence, inhibition of hLDH-5 may constitute an efficient way to interfere with tumour growth and invasiveness. This review provides an overview of the LDH inhibitors that have been developed up to now, an analysis of their possible isoform-selectivity, and their therapeutic potentials.

QSAR models for predicting enzymatic hydrolysis of new chemical entities in 'soft-drug' design.

The work described here is aimed at developing QSAR models capable of predicting in vitro human plasma lability/stability. They were built based on a dataset comprising about 200 known compounds. 3D structures of the molecules were drawn, optimized and submitted to the calculation of molecular descriptors that enabled selecting different TR/TS set pairs, subsequently exploited to develop QSAR models. Several 'machine learning' algorithms were explored in order to obtain suitable classification models, which were then validated on the relevant TS sets. Moreover the predictive ability of the best performing models was assessed on a Prediction set (PS) comprising about 40 molecules, not strictly related, from a structural point of view, to the initial dataset, but (obviously) comprised within the validity domain of the QSAR models obtained. The study allowed selecting predictive models enabling the classification of New Chemical Entities with regard to hydrolysis rate, that may be exploited for soft-drug design.

Metabolically labile cannabinoid esters: a 'soft drug' approach for the development of cannabinoid-based therapeutic drugs.

Biphenylic ester derivatives, designed by using a 'soft-drug' approach, proved to possess good binding properties toward cannabinoid CB(1) and CB(2) receptors and, at the same time, their metabolically labile ester portion would promote a rapid systemic inactivation. This may constitute a possible solution to the psychotropic side effects encountered when cannabinoids are therapeutically employed as local analgesic or antiglaucoma agents.

Resveratrol structure and ceramide-associated growth inhibition in prostate cancer cells.

Resveratrol (3,4',5-trans-trihydroxystilbene) is a dietary polyphenol with chemopreventive properties present in grapes, red wine, peanuts and other edible products. The antiproliferative and proapoptotic effect of resveratrol in breast cancer cells can be traced to the accumulation of ceramide. In this study we demonstrate that resveratrol can also exert antiproliferative/proapoptotic effects in association with the accumulation of endogenous ceramide in the androgen receptor (AR)-negative prostate cancer cell line, PC3. Notably, resveratrol shares with other ceramide-inducing agents a phenolic moiety on its structure. For this reason we hypothesize that the phenolic moiety is critical for the ceramide-associated growth-inhibitory effects of resveratrol. We compared the ability to induce both ceramide increase and growth inhibition in PC3 cells of resveratrol and three resveratrol analogs: piceatannol (3,3',4',5-trans-tetrahydroxystilbene), with an additional hydroxyl group in the 3' position; trans-stilbene, the nonhydroxylated analog; and the semisynthetic 3,4',5-trimethoxy-trans-stilbene (TmS), with methoxyl groups in lieu of the hydroxyl groups. Of the three stilbenoids, only piceatannol (and not stilbene or TmS) produced ceramide-associated growth inhibition. These data point to the phenolic moiety of stilbenoids as a critical structural feature necessary to induce ceramide-associated growth inhibition.

7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Salicylaldoxime moiety as a phenolic "A-Ring" substitute in estrogen receptor ligands.

The phenolic "A-ring" of natural and synthetic estrogen receptor (ER) ligands was effectively replaced by a planar six-member ring formed through an intramolecular hydrogen bond within a salicylaldoxime. Thus, oxime 1, a structural analogue of a triarylethylene estrogen, showed a significant binding affinity for the ER. The OH of the oxime function appears to mimic the phenolic OH present in more "classical" ER ligands because the binding reduced when the oxime OH is methylated (2) or absent (3).

Design, synthesis, and characterization of the antitumor activity of novel ceramide analogues.

A deficiency in apoptosis is one of the key events in the proliferation and resistance of malignant cells to antitumor agents; for these reasons, the search for apoptosis-inducing drugs represents a valuable approach for the development of novel anticancer therapies. In this study we report the first example of conformationally restrained analogues of ceramide (compounds 1-4), where the polar portion of the molecule has been replaced by a thiouracil (1, 3) or uracil (2, 4) ring. The evaluation of their biologic activity on CCRF-CEM human leukemia cells demonstrated that the most active was compound 1 followed by compound 2 (mean 50% inhibition of cell proliferation [IC(50)] 1.7 and 7.9 microM, respectively), while compounds 3 and 4 were inactive, as were uracil, thiouracil, and 5,6-dimethyluracil, the pyrimidine moieties of compounds 1-4. For comparison, the IC(50) of the reference substance, the cell-permeable C2-ceramide, was 31.6 microM. Compounds 1 and 2 and C2-ceramide were able to trigger apoptosis, as shown by the occurrence of DNA and nuclear fragmentation, and to release cytochrome c from treated cells. The treatment of female CD-1 nu/nu athymic mice bearing a WiDr human colon xenograft with the most active compound 1 at 2, 10, 50, and 200 mg/kg ip daily for 10 days resulted in an antitumor effect that was equivalent at 50 mg/kg or superior (200 mg/kg) to that of cyclophosphamide, 20 mg/kg ip daily, delivered on the same schedule, with markedly lower systemic toxicity. In conclusion, the present study demonstrates that the new ceramide analogues 1 and 2 are characterized by in vitro and in vivo antitumor activity and low toxicity.

Use of beta-cyclodextrin in the capillary zone electrophoretic separation of the components of clandestine heroin preparations.

The present paper describes the methodological optimization and validation of a capillary zone electrophoresis method for the rapid determination of heroin, secondary products and additives present in clandestine heroin samples, by using 20 mM beta-cyclodextrins in phosphate buffer, pH 3.23. Applied potential was 15 kV and separation temperature was 24 degrees C; detection was by UV absorption at 200 nm wavelength. Heroin samples were first dissolved in CHCl3-MeOH (96:4, v/v) and injected by pressure (0.5 p.s.i., 3 s; 1 p.s.i.=6894.76 Pa) after evaporation of the organic mixture and reconstitution in aqueous buffer. Under the described conditions, phenylethylamine (internal standard), morphine, monoacetylmorphine, heroin, acetylcodeine, papaverine, codeine and narcotine were baseline resolved in less than 10 min. The limit of detection was better than 1 microg/ml for each analyte. The study of the intra-day and day-to-day precision showed, in terms of migration times, RSDs < or = 0.71% and, in terms of peak areas, RSDs < or = 3.2%. Also, the evaluation of linearity and analytical accuracy of the method provided good results for all the analytes investigated, thus allowing its application to real cases of seized controlled drug preparations.

Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells.

Proteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC(50)) of 0.47 +/- 0.03 and 5.24 +/- 1.41 microM (mean +/- SE), respectively. The isobologram analysis performed at the IC(50)level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras.

Synthesis and inhibitory activity towards human leukocyte elastase of new 7alpha-methoxy and 7alpha-chloro (2-acyloxymethyl) cephem derivatives.

Some new cephem derivatives of types 4 and 5, viewed as analogues of type I esters in which the atomic sequence of the C-2 ester group is formally inverted, were synthesised and tested in vitro for their inhibitory activity towards human leukocyte elastase and porcine pancreatic elastase. An examination of the inhibition data obtained for the new type 4 and 5 derivatives, while exhibiting a considerable reduction in their activity against porcine pancreatic elastase, indicated that these compounds still maintain an appreciable inhibitory activity against human leukocyte elastase. On this basis the new type of C-2 substitution appears to contribute to the research of new, potentially interesting, cephalosporinic human leukocyte elastase inhibitors.

Synthesis and dopaminergic properties of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine, a potent and selective dopamine D4 receptor ligand.

The synthesis of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine 1, a potent and selective D4 dopaminergic ligand, was performed. The 3-(3,4-dimethylphenyl)- 1-propylpiperidine with the R configuration showed an affinity for the D4 receptors 6-fold higher than the corresponding enantiomer with the S configuration. Furthermore, the (R)-1 enantiomer proved to be highly selective for D4 receptors with respect to D2-D3 receptors, with a Ki ratio higher than 25,000, while the (S)-1 enantiomer was about 100-fold less selective than the (R)-1 one.

Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.