Climate change projected effects on coastal foundation communities of the Greater Everglades using a 2060 scenario: need for a new management paradigm.

Rising sea levels and temperature will be dominant drivers of coastal Everglades' foundation communities (i.e., mangrove forests, seagrass/macroalgae, and coral reefs) by 2060 based on a climate change scenario of +1.5 °C temperature, +1.5 foot (46 cm) in sea level, ±10 % in precipitation and 490 ppm CO2. Current mangrove forest soil elevation change in South Florida ranges from 0.9 to 2.5 mm year(-1) and would have to increase twofold to fourfold in order to accommodate a 2060 sea level rise rate. No evidence is available to indicate that coastal mangroves from South Florida and the wider Caribbean can keep pace with a rapid rate of sea level rise. Thus, particles and nutrients from destabilized coastlines could be mobilized and impact benthic habitats of southern Florida. Uncertainties in regional geomorphology and coastal current changes under higher sea levels make this prediction tentative without further research. The 2060 higher temperature scenario would compromise Florida's coral reefs that are already degraded. We suggest that a new paradigm is needed for resource management under climate change that manages coastlines for resilience to marine transgression and promotes active ecosystem management. In the case of the Everglades, greater freshwater flows could maximize mangrove peat accumulation, stabilize coastlines, and limit saltwater intrusion, while specific coral species may require propagation. Further, we suggest that regional climate drivers and oceanographic processes be incorporated into Everglades and South Florida management plans, as they are likely to impact coastal ecosystems, interior freshwater wetlands and urban coastlines over the next few decades.

Inactivation of lipid enveloped viruses by octanoic Acid treatment of immunoglobulin solution.

Inactivation of lipid enveloped viruses by treatment with octanoic acid has been investigated for three intravenous immunoglobulin preparations, using Human Immunodeficiency Virus, Bovine Viral Diarrhoea Virus, Sindbis Virus and Pseudorabies Virus as test viruses. At a concentration of 7.45 g octanoic acid per kg solution complete inactivation of lipid enveloped viruses to below detectable level (>5.36, >4.68, >6.25 and >5.55 log(10), respectively) was achieved within the first minutes of treatment. Octanoic acid treatment as described here, has been demonstrated as an effective and rapid virus inactivation procedure, which shows high robustness at the tested ranges of temperature, pH and protein content of the test material. However, pH must be considered as a critical parameter of treatment, as octanoic acid fails to inactivate lipid coated viruses at basic pH. At suitable conditions, e.g. pH<6.0 and a concentration of >3.7 g/kg, octanoic acid treatment gives reliable and highly effective inactivation of lipid enveloped viruses.

Radially expanding laparoscopic access for renal/adrenal surgery.

OBJECTIVES: To use a radially expanding system (Step) and a modified port location for intra-abdominal access to decrease the access-related complications in renal and adrenal surgery. Access-related complications during laparoscopic renal surgery are frustrating and are more common in patients with previous abdominal surgery and associated adhesions. METHODS: Laparoscopic upper tract procedures were performed in 62 patients using radially expanding trocars, and the results were reviewed with regard to access, port placement, and associated complications. For initial access, a Veress needle was placed subcostally in the midclavicular line. An expandable mesh sleeve trocar was used for trocar insertion after a pneumoperitoneum was established. A blunt-tipped fascial dilator was used to dilate to 10 or 12 mm. Additional ports were placed in an L shape (nephrectomy) or a subcostal configuration (adrenalectomy) under direct vision using the Step ports. RESULTS: Of 62 patients, 24 had had prior abdominal surgery. Open insertion of the mesh sleeve was necessary in 20%, of whom 60% had had prior abdominal surgery. In 9% of cases, the liver was punctured with the initial pass of the Veress needle. Only minimal bleeding from the injury site was noticed. The liver punctures did not require cauterization and did not result in conversion to an open procedure. At a mean follow-up of 12 months, no access-related complications or port-site hernias were noted. CONCLUSIONS: Placement of the initial access subcostally at the level of the midclavicular line helps to prevent visceral injury, especially in patients with previous abdominal surgery. The use of the radially expanding access system with the modification of port location allows safe and rapid laparoscopic access for upper urinary tract surgery. This trocar system is an excellent alternative to the standard laparoscopic trocars.

Radially expanding single-step nephrostomy tract dilator.

OBJECTIVES: To compare the radial and axial forces produced by balloon, Amplatz, and radially expanding single-step nephrostomy (RESN) systems and report our initial clinical results using the new RESN device. Balloon, Amplatz, and Alken dilators are commonly used to establish nephrostomy tracts in percutaneous surgery. They require multiple steps, with the potential for kinking and displacement of the working guidewire. In contrast, the new RESN tract dilator expands a unique sleeve conduit and places an Amplatz-like sheath in a single step with less dependence on a guidewire for dilation. METHODS: An experimental model was designed using a perforated silicon disc with a 10F central opening to measure the axial force transmission as 30F balloon, Amplatz, and RESN systems were inserted through the silicon discs. We also report our first 9 patients who underwent percutaneous dilation with the RESN system. RESULTS: Thirty French expansion was achieved with each dilator tested. Substantially lower axial forces were transmitted with the RESN device compared with the balloon and Amplatz dilators (5.2 versus 13.1 and 19.2 lb, respectively, P <0.001). Intraoperatively, all 9 patients were successfully dilated, and the kidney was relatively stationary as imaged with fluoroscopy. One patient with multiple prior renal procedures was successfully dilated with RESN system after failed attempts with balloon dilation. CONCLUSIONS: The RESN dilator is a rapid, single-step access system successfully used in our first 9 patients. Intraluminal sleeve dilation eliminates guidewire dependence for maintaining access, limits renal displacement, and facilitates appropriate vector force for percutaneous dilation.

The laparoscopic cigarette sponge.

PURPOSE: Minor hemorrhage during laparoscopic procedures may obscure the operative field. We describe the use of an especially designed, 4 x 4 absorbent sponge for multiple laparoscopic applications. MATERIALS AND METHODS: The cigarette sponge, also known as the Kittner roll gauze, was routinely used for laparoscopic upper tract procedures. The sponge may be placed easily through ports 5 mm. or greater. RESULTS: The cigarette sponge was excellent for absorbing minor but bothersome bleeding, facilitating suction and blunt dissection, and assisting with retraction. CONCLUSIONS: This especially designed laparoscopic sponge dramatically eases laparoscopic procedures, especially for controlling bothersome hemorrhage and blunt dissection. It may decrease operative time and facilitate difficult laparoscopic procedures.

Prostaglandins are required for CREB activation and cellular proliferation during liver regeneration.

The liver responds to multiple types of injury with an extraordinarily well orchestrated and tightly regulated form of regeneration. The response to partial hepatectomy has been used as a model system to elucidate the molecular basis of this regenerative response. In this study, we used cyclooxygenase (COX)-selective antagonists and -null mice to determine the role of prostaglandin signaling in the response of liver to partial hepatectomy. The results show that liver regeneration is markedly impaired when both COX-1 and COX-2 are inhibited by indocin or by a combination of the COX-1 selective antagonist, SC-560, and the COX-2 selective antagonist, SC-236. Inhibition of COX-2 alone partially inhibits regeneration whereas inhibition of COX-1 alone tends to delay regeneration. Neither the rise in IL-6 nor the activation of signal transducer and activator of transcription-3 (STAT3) that is seen during liver regeneration is inhibited by indocin or the selective COX antagonists. In contrast, indocin treatment prevents the activation of CREB by phosphorylation that occurs during hepatic regeneration. These data indicate that prostaglandin signaling is required during liver regeneration, that COX-2 plays a particularly important role but COX-1 is also involved, and implicate the activation of CREB rather than STAT3 as the mediator of prostaglandin signaling during liver regeneration.

Retrograde renoscopic fragmentation of moderate-size (1.5-3.0-cm) renal cystine stones.

PURPOSE: We present a series of cystinuric patients with renal cystine calculi between 1.5 and 3.0 cm treated with retrograde renoscopy and intracorporeal lithotripsy and report our results, complications, and inpatient utilization with this approach. PATIENTS AND METHODS: The hospital and office charts of five consecutive patients with six treated renal units who underwent retrograde renoscopy and electrohydraulic lithotripsy for renal cystine stones between 1.5 and 3.0 cm were reviewed. Data on stone size and location, procedures performed, results, complications, and inpatient hospital days were compiled. RESULTS: Five of the six renal units were either rendered stone free or had fragments totalling 3 mm or less. Three renal units required only a single procedure, one required repeat ureteroscopy for Steinstrasse, and one required SWL and repeat ureteroscopy for Steinstrasse. One renal unit was left with a 6-mm fragment for which the patient refused further treatment. There were no major complications. The mean hospital stay was 1 day, and the mean number of procedures per patient was 1.3. CONCLUSION: Retrograde renoscopy and intracorporeal lithotripsy for renal cystine stones 1.5 to 3.0 cm is safe and effective and should be considered as an alternative to percutaneous nephrolithotomy in these patients.

The effect of intraocular pressure on human and rabbit scleral permeability.

PURPOSE: The purpose of this study was to evaluate the effects of intraocular pressure on the permeability of human and rabbit sclera to water, dexamethasone, and carboxyfluorescein. METHODS: Scleral sections excised from moist-chamber-stored human globes or eyes obtained from euthanatized New Zealand White rabbits were mounted in a perfusion chamber that can create a transscleral pressure that simulates an intraocular pressure. A small depot of drug (100 microl) was added to the episcleral surface while perfusing an irrigating solution slowly across the choroidal side. The perfusate was collected and scleral permeability calculated. Experiments were performed at 0, 15, 30, and 60 mm Hg for each compound in human and rabbit tissue. RESULTS: Analysis of variance showed a significant effect of intraocular pressure on both human and rabbit scleral permeability. Human scleral permeability was decreased by as much as a factor of two for water (P = 0.0004), dexamethasone (P<0.0001), and carboxyfluorescein (P = 0.0064) at elevated intraocular pressures. Rabbit scleral permeability was similarly affected by elevated intraocular pressure for water (P = 0.0039), dexamethasone (P = 0.0001), and carboxyfluorescein (P = 0.0016). CONCLUSIONS: This study shows that simulated intraocular pressure ranging from 15 to 60 mm Hg can decrease scleral permeability to small molecules by one half when compared with the sclera with no pressure applied.

Intracameral anesthesia: in vitro iris and corneal uptake and washout of 1% lidocaine hydrochloride.

OBJECTIVES: To characterize the uptake, washout, and metabolism of lidocaine hydrochloride in the iris/ciliary body and cornea. METHODS: Iris/ciliary body uptake of lidocaine hydrochloride was measured by incubating human and rabbit irides in radiolabeled carbon 14-1% lidocaine hydrochloride for 2 to 60 minutes. Washout was determined by incubating the iris in 14C-1% lidocaine hydrochloride for 5 minutes and transferring the iris to a series of wells. The wells contained a common intraocular irrigating solution of essential ions, glucose, and glutathione buffered with bicarbonate (an enriched balanced salt solution [BSS PLUS]), which is similar to aqueous humor. Corneal uptake was measured by exposing the endothelial surface to 14C-1% lidocaine hydrochloride for 5 or 15 minutes. Corneal washout was performed after 5-minute exposure to 14C-1% lidocaine hydrochloride using a 2-chambered diffusion apparatus. Samples of the iris, cornea, and BSS PLUS washout solution were analyzed by high-performance liquid chromatography and liquid scintillation spectrometry. RESULTS: In vitro iris/ciliary body uptake of 14C-1% lidocaine hydrochloride follows a logarithmic curve, with 50% to 60% of maximum lidocaine hydrochloride uptake present at 5 minutes. There was no difference in uptake between human, albino rabbit, and pigmented rabbit irides. Washout of lidocaine from the iris occurs with a halflife of 8 to 9 minutes. Corneal uptake of lidocaine was greater after incubation for 15 vs. 5 minutes. The washout of lidocaine from the cornea had a half-life of 5 minutes. Results of high-performance liquid chromatography confirmed that there were no metabolites or breakdown products in the iris, cornea, or washout solution. CONCLUSIONS: Lidocaine is taken up quickly by the iris/ ciliary body and cornea and rapidly removed from these tissues after BSS PLUS washout. Irrigation during phacoemulsification seems to limit lidocaine exposure to the ocular tissues, resulting in a short duration of anesthesia. Lidocaine is not metabolized or broken down by the iris or cornea during this short period.

Improvement of virus safety of a S/D-treated factor VIII concentrate by additional dry heat treatment at 100 degrees C.

In order to increase the virus safety of a solvent/detergent-treated Factor VIII concentrate in regard to non-lipid coated viruses and to respond to the continuous discussion about reports on hepatitis A transmission by Factor VIII preparations, we have investigated the effect of a terminal dry heat treatment (30 min 100 degrees C) on HAV and various other viruses. By this treatment Hepatitis A virus was inactivated below detectable level after a few minutes (> 5.3 log10). Other RNA viruses such as the Human Immunodeficiency Virus (> 6.6 log10), bovine viral diarrhoea virus (> 6.6 log10) and vesicular stomatitis virus (> 5.8 log10) were also inactivated below detectable level. Pseudo rabies virus and reovirus Type 3 are inactivated by 5.7 and > 6.0 log10, respectively. SV40 and bovine parvo virus showed significant resistance to dry heat treatment. We conclude that the involvement of two strong virus inactivation steps, acting by different mechanisms, improves the virus safety of Factor VIII concentrates without destroying the Factor VIII activity. Moreover, the terminal 100 degrees C heat treatment for 30 min represents an effective measure to inactivate non-lipid enveloped viruses, in particular hepatitis A, which is resistant to solvent/detergent treatment.

Validation of virus inactivation and removal for the manufacturing procedure of two immunoglobulins and a 5% serum protein solution treated with beta-propiolactone.

Intravenous immunoglobulins and serum protein solutions are manufactured from human plasma pools of healthy, screened donors. A step-by-step validation of virus removal and/or inactivation was performed for the manufacturing process, which includes cold ethanol fractionation, beta-propiolactone (beta-PL) treatment, UV irradiation, thermal inactivation and other chemical and physical purification steps. The total viral clearance factors achieved for the entire manufacturing process were by several magnitudes greater than the potential virus load of current plasma pools. Human immunodeficiency virus 1 (HIV-1) infectivity was reduced by > 13.4 log for 7S immunoglobulin, > 15.3 log for IGM enriched immunoglobulin and > 16 log for a 5% serum protein solution. In addition, high clearance rate for a broad spectrum of model viruses was demonstrated for all three blood derivatives being > 23.2 to > 27.8 log for pseudo rabies virus (PSR), > 12.3 to > 22.6 log for vesicular stomatitis virus (VSV) and 6.9-10.6 log for simian virus 40 (SV40). For the beta-propiolactone inactivation step Hepatitis C model viruses, e.g. equine arteritis virus (EAV) and bovine viral diarrhoea virus (BVDV) were also investigated.

Use of photoactivatable peptide substrates of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) to characterize a myristoyl-CoA-Nmt1p-peptide ternary complex and to provide evidence for an ordered reaction mechanism.

Nmt1p (EC 2.3.1.97) catalyzes the transfer of myristate (C14:0) from coenzyme A to the N-terminal glycine residue of a variety of eukaryotic cellular and viral proteins. Our recent studies of the 455-amino acid Saccharomyces cerevisiae acyltransferase (Nmt1p) suggested that its mechanism of catalysis is ordered Bi Bi with myristoyl-CoA binding occurring prior to binding of peptide and release of CoA occurring prior to release of the myristoyl-peptide. The interaction between enzyme and peptide has now been examined in greater detail by using photoactivatable octapeptide substrates containing 125I-labeled azidosalicyclic acid attached via an amide bond to the gamma-amino group of a diaminobutyrate residue located at position 2 or the epsilon-amino group of a lysine residue located at position 8. The photopeptides can be specifically crosslinked to chymotryptic fragments of Nmt1p in the presence but not in the absence of a nonhydrolyzable myristoyl-CoA analog, S-(2-oxo)pentadecyl-CoA. Labeling of the chymotryptic fragments is markedly reduced when GLYASKLS, a high-affinity substrate derived from residues 2-9 of S. cerevisiae ADP-ribosylation factor 2, or ALYASKLS, a competitive inhibitor (for peptide), is added with the iodinated photopeptide. These findings suggest that peptide affinity for the acyl-CoA-Nmt1p binary complex is much greater than it is for apoNmt1p, consistent with the ordered Bi Bi mechanism ascribed to Nmt1p. Finally, automated sequential Edman degradation of these chymotryptic fragments suggests that the peptide binding domain of Nmt1p may be composed of elements from two protease-resistant domains, Arg42-Try219 and Thr220-Leu455.

Genetic and biochemical studies of a mutant Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase, nmt72pLeu99-->Pro, that produces temperature-sensitive myristic acid auxotrophy.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that transfers myristate from CoA to the amino-terminal glycine residue of at least 12 cellular proteins. Its reaction mechanism is Ordered Bi Bi with myristoyl-CoA binding occurring before binding of nascent polypeptides and release of CoA preceding release of the myristoylprotein product. nmt1-72 is a temperature-sensitive allele, identified by Stone et al. (Stone, D. E., Cole, G. M., Lopes, M. B., Goebl, M., and Reed, S. I. (1991) Genes & Dev. 5, 1969-1981) that causes arrest in the G1 phase of the cell cycle due to reduced acylation of Gpa1p. We have recovered this mutant allele and determined that it contains a single point mutation resulting in a Leu99 (CTA) to Pro (CCA) substitution. Addition of > or = 500 microM myristate but not palmitate to synthetic or rich media rescues the growth arrest caused by nmt1-72 at 37-39 degrees C, consistent with the observation that purified nmt72p has reduced affinity for myristoyl-CoA and that exogenous myristate but not palmitate increases cellular myristoyl-CoA pools. Metabolic labeling studies in S. cerevisiae and co-expression of nmt72p with several protein substrates of Nmt1p in Escherichia coli indicate that the Leu99-->Pro substitution causes a reduction in the acylation of some but not all protein substrates. Since formation of a myristoyl-CoA.Nmt1p complex appears to be required for synthesis/formation of a peptide binding site, these defects in acylation appear to arise either because Leu99 is a component of the enzyme's functionally distinguishable myristoyl-CoA and peptide recognition sites or because Pro99 alters the interaction between myristoyl-CoA and enzyme in a way that precludes formation of a normal peptide binding site. The reduction in affinity for myristoyl-CoA produced by Leu99-->Pro in nmt72p is less than that produced by the Gly451-->Asp mutation in nmt181p, which also produces temperature-sensitive myristic acid auxotrophy. Isogenic, haploid strains containing NMT1, nmt1-72, and nmt1-181 do not manifest any obvious differences in steady state levels of the acyltransferases during growth at permissive temperatures or in the biosynthesis of long chain saturated acyl-CoAs. The spectrum of cellular N-myristoylproteins whose level of acylation is affected by nmt1-72 and nmt1-181 is distinct.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary to maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing Nmt1p or its mutant derivatives and one of two eukaryotic substrates for the wild type enzyme (S. cerevisiae Gpa1p and rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, and monitoring Nmt-dependent incorporation of exogenous [3H]myristate into the G protein alpha subunits or (ii) an in vitro enzyme assay using lysates prepared from bacteria producing wild type or mutant Nmts. The data indicate that the minimal catalytic domain of Nmt1p is located between Ile59-->Phe96 and Gly451-->Leu455. Analyses of the ability of mutant nmtps to rescue the lethal phenotype of an nmt1 null allele in a haploid strain of yeast grown on rich media, with or without blockade of cellular fatty acid synthetase, suggest that the amino-terminal 59 residues of Nmt1p may play an important noncatalytic role, functioning as a targeting signal so this cytosolic enzyme can access cellular myristoyl-CoA pools generated from activation of exogenous C14:0 by acyl-CoA synthetase(s). Moreover, there appear to be differences in the location or accessibility of myristoyl-CoA pools derived from fatty acid synthetase and acyl-CoA synthetases. The E. coli co-expression system was used to map structural elements that determine differences in the peptide substrate specificities of Nmt1p and the orthologous human Nmt. Rat Go alpha is a substrate for both enzymes, whereas human Gz alpha is a substrate only for human NMT. Studies of a series of chimeric enzymes composed of elements from the amino- or carboxyl-terminal portions of human and yeast Nmts indicate that (i) recognition/utilization of Gz alpha involves elements distributed from the amino-terminal half through the region defined by Leu352-->Lys410 of the 416 residue human enzyme and (ii) formation of a fully functional peptide binding site and a fully functional myristoyl-CoA binding site in either of these enzymes requires contributions from both their amino-terminal and carboxyl-terminal halves.

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active substrates for the mutant enzyme as myristoyl-CoA at 24 degrees C. However, unlike C16:0, they produce growth arrest of nmt181p-producing cells at this "permissive" temperature, suggesting that these C16 fatty acids do not allow expression of the biological functions of essential S. cerevisiae N-myristoylproteins.

Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.

Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees. The activities of NMT's acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives. Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli.

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.