Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

Cuticle characteristics and volatile emissions of petals in Antirrhinum majus

Floral volatiles, which are small and generally water-insoluble, must move from their intracellular sites of synthesis through the outermost cuticle membrane before release from the flower surface. To determine whether petal cuticle might influence volatile emissions, we performed the first analysis of petal cuticle development and its association with the emission of flower volatiles using Antirrhinum majus L. (snapdragon) as a model system. Petal cuticular wax amount and composition, cuticle thickness and ultrastructure, and the amounts of internal and emitted methylbenzoate (the major snapdragon floral scent compound) were examined during 12 days, from flower opening to senescence. Normal ( n -) alkanes were found to be the major wax class of snapdragon petals (29.0% to 34.3%) throughout the 12 days examined. Besides n -alkanes, snapdragon petals possessed significant amounts of methyl branched alkanes (23.6–27.8%) and hydroxy esters (12.0–14.0%). Hydroxy esters have not been previously reported in plants. Changes in amount of methylbenzoate inside the petals followed closely with levels of methylbenzoate emission, suggesting that snapdragon petal cuticle may provide little diffusive resistance to volatile emissions. Moreover, clear associations did not exist between methylbenzoate emission and the cuticle properties examined during development. Nevertheless, the unique wax composition of snapdragon petal cuticles shows similarities with those of other highly permeable cuticles, suggesting an adaptation that could permit rapid volatile emission by scented flowers.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.     

Design,synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH₂ group, inhibits HDHD4 with a binding affinity (IC₅₀ 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, K_m 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg²⁺. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca²⁺ and Mg²⁺ are indicative of a highly dynamic process in the active site for the HDHD4/Mg²⁺/3 complex. Possible explanations for this observation are discussed.     

Polyamines in Human Brain: Regional Distribution and Influence of Aging

Abstract: Depolarization of adult rat forebrain slices with veratrine induced the release of excitatory amino acids (glutamate and aspartate), the synthesis of nitric oxide (NO), and increases in cyclic GMP (cGMP). The NO synthase inhibitors N ^ω-monomethyl- l -arginine and N ^ω-nitro- l -arginine methyl ester decreased the release of NO and the levels of cGMP without affecting the release of excitatory amino acids. In contrast, the antiepileptic drug lamotrigine inhibited the release of excitatory amino acids and of NO, and decreased the levels of cGMP without causing a significant direct inhibition of the NO synthase. Furthermore, the synthesis of NO and the increases in cGMP induced by veratrine were partially blocked by the N -methyl- d -aspartate (NMDA) receptor antagonist MK-801 but not by 6-nitro-7-sulphamobenzo( f )quinoxaline-2,3-dione, a non-NMDA receptor antagonist. Neither of these compounds inhibited directly the NO synthase or the release of excitatory amino acids. Thus, these three types of compound act as an inhibitor of voltage-sensitive sodium channels (lamotrigine), as a receptor antagonist (MK-801), or as direct inhibitors of the NO synthase, to block the pathway leading to increased cGMP after veratrine depolarization. It is likely that some of the pharmacological and therapeutic actions shared by these three types of compound are, at least in part, a consequence of inhibition of the synthesis of NO.     

Potent and selective biphenyl azole inhibitors of adipocyte fatty acid binding protein (aFABP)

Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.     

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site

Aims: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non‐point source subtropical marine beach. Methods and Results: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Conclusions: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin‐resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non‐point source beaches. Significance and Impact of the Study: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non‐point sources beaches.