The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

Biotechnology applications of amino acids in protein purification and formulations

Summary. Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called “compatible solutes”, since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein–protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.     

Abnormal changes of plasma acute phase proteins in schizophrenia and the relation between schizophrenia and haptoglobin (Hp) gene

Summary. In this study we focused on detecting schizophrenia related changes of plasma proteins using proteomic technology and examining the relation between schizophrenia and haptoglobin (Hp) genotype. We investigated plasma proteins from schizophrenic subjects (n = 42) and healthy controls (n = 46) by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry. To further reveal the genetic relationship between acute phase proteins (APPs) and schizophrenia disease, we tested Hp α1/Hp α2 (Hp 1/2) polymorphism and two single nucleotide polymorphisms (SNPs) of Hp, rs2070937 and rs5473, for associations with schizophrenia in the Chinese Han population. With the relatively high number of samples for 2-DE work, we found that four proteins in the family of positive APPs were all up-regulated in patients. In genetic association study, we found significant associations existing between schizophrenia and Hp polymorphisms, Hp 1/2 and rs2070937 variants. Schizophrenia is accompanied by both an altered expression of Hp protein and a different genotype distribution of Hp gene, demonstrating that Hp is associated with schizophrenia. The results from proteomic and genomic aspects both indicate that acute phase reaction is likely to be an aetiological agent in the pathophysiology of schizophrenia, but not just an accompanying symptom. The positive APPs are schizophrenic related proteins, with the highly concordant results on four positive APPs. The first two authors contributed equally.     

High-throughput capillary electrophoresis method for plasma cysteinylglycine measurement: evidences for a clinical application

Summary. Increased levels in plasma homocysteine and cysteine, and more recently, decreased levels in cysteinylglycine have been indicated as a risk factor for vascular diseases. Most assays focused their attention only on homocysteine determination and when also other thiols were measured, analytical times drastically increased. By modifying our previous method for thiols detection, we set up a rapid capillary electrophoresis method for the selective quantification of plasma cysteinylglycine, cutting the analysis time of about 50%. Samples were treated with tri-n-butylphosphine as reducing agent, proteins were precipitated with trichloroacetic acid and released thiols were successively derivatized by the selective thiol laser-induced fluorescence-labeling agent 5-iodoacetamidofluorescein and separated by capillary electrophoresis. A baseline separation between peaks was obtained in about 2 min using 3 mmol/L sodium phosphate/2.5 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.25 in a 47 cm long capillary with a cartridge temperature of 45 °C. The method application was checked by measuring plasma Cys-Gly levels in a group of patients affected by retinal vein occlusion (RVO), an important cause of visual loss in the elderly. The low levels of Cys-Gly found in the RVO patients suggest that these small thiols may have importance in the disease development. Authors’ addresses: Dr. Angelo Zinellu, Dr. Ciriaco Carru, Department Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy     

Structural properties of proteins specific to the myelin sheath

Summary. The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small number of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a number of neurological diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a number of myelin-specific proteins are described. Author’s address: Dr. Petri Kursula, Department of Biochemistry, University of Oulu, FIN-90014 Oulu, Finland     

Depletion of the high-abundance plasma proteins

Summary. Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.Current address: Foundation for Biomedical Research of the Academy of Athens, Greece     

Structural diversity of bicyclic amino acids

Summary. Over the years biomedical research has been constantly oriented towards the development of new therapeutics based on bioactive peptides and their analogues. In particular, the generation of compounds having structures and functions similar to bioactive peptides, named “peptidomimetics”, raised much interest among organic and medicinal chemists due to the possibility by using such compounds to improve both potency and stability of peptidic lead compounds. In the context of this research area, unnatural amino acids are of great interest in drug discovery, and their use as new building blocks for the development of peptidomimetics with high diversity level and possessing high-ordered structures is of special interest. In particular, medicinal chemistry has taken advantage of the use of amino acid homologues and of cyclic and polycyclic templates to introduce elements of diversity for the generation of new molecules as drug candidates. Bicyclic amino acids have been developed as reverse turn mimetics and dipeptide isosteres, and the constraint imposed by their structures has been reported as a tool for controlling the conformational preferences of modified peptides. Moreover, synthetic efforts have been driven to the generation of diverse structures based on the modulation of ring size and scaffold decoration by suitable functional groups. Herein is reported an overview of different classes of bicyclic amino acids, taking into account the strategies to achieve structurally diverse templates, and some implications in medicinal chemistry are also disclosed. Authors’ address: Antonio Guarna, Dipartimento di Chimica Organica “Ugo Schiff” and Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Bioattivi (HeteroBioLab), Università degli Studi di Firenze, Polo Scientifico e Tecnologico, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Firenze, Italy     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

S-adenosylmethionine and its products

Summary. S-adenosylmethionine is involved in many processes, mainly methylation, polyamine synthesis and radical-based catalysis. It is synthesised through the catalysis of differently regulated enzyme forms. When it is used, the compounds formed are reutilized in different ways: in case of methylation, its end product is homocysteine, which can be remethylated to methionine, give rise to cysteine in the so-called transsulphuration pathway, or be released; in the case of polyamine synthesis, the methylthioadenosine formed is cleaved and gives rise to compounds which can be reutilized; during radical-based catalysis, 5-deoxyadenosine is formed and this, too, is cleaved and reutilized. Authors’ address: Prof. M. A. Grillo Dipartimento di Medicina e Oncologia Sperimentale, Biochimica, Università di Torino, Torino, Italy     

Indicators of l-arginine metabolism and cardiovascular risk factors A cross-sectional study in healthy middle-aged men

Summary. This study examines the relationship between traditional risk factors of coronary artery disease and indicators involved in the metabolism of l-arginine (plasma and urine l-arginine, plasma l-citrulline, serum creatinine and urine orotic acid). Our study population consisted of 40 healthy male volunteers aged between 35 and 55 years. We found an inverse association between serum creatinine and blood pressure, between plasma l-citrulline and blood pressure, as well as between urine l-arginine and blood pressure. We also found a positive association between plasma LDL-cholesterol and urine l-arginine and a negative correlation between plasma l-arginine and LDL-cholesterol. Orotic acid measured from urine was not associated with any of the indicators of l-arginine metabolism. Our results indicate that l-arginine metabolism is of profound significance for cardiovascular health. However, our study does not answer questions relating to causality. Further studies are needed to clarify the causal relationship between cardiovascular risk factors, especially elevated blood pressure and high LDL-cholesterol, and indicators of l-arginine metabolism. Received January 18, 1999     

Strategies to improve plasma half life time of peptide and protein drugs

Summary. Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half life times. By shortening the overall amino acid amount of somatostatin and replacing l-analogue amino acids with d-amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin. A PEG_2,40 K conjugate of INF-α-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

Synthesis of a versatile constrained analogue of dipeptide DG (Asp-Gly)

Summary. The synthesis of an orthogonally protected constrained analogue of dipeptide DG (Asp-Gly) is reported exploiting alkylation of a chiral lactam. The versatility of this analogue was proven by removal of t-Boc protecting group, followed by coupling under homogeneous conditions with t-Boc-Arg(Z₂)-Gly, to give a conformationally restricted analogue of RGDG tetrapeptide. Authors’ address: Mario Orena, Dipartimento di Scienze e Tecnologie Chimiche, Università Politecnica delle Marche, Via Brecce Bianche, I-60131 Ancona, Italy     

Virtual screening for finding natural inhibitor against cathepsin-L for SARS therapy

Summary. Recently Simmons et al. reported a new mechanism for SARS virus entry into target cells, where MDL28170 was identified as an efficient inhibitor of CTSL-meditated substrate cleavage with IC₅₀ of 2.5 nmol/l. Based on the molecule fingerprint searching method, 11 natural molecules were found in the Traditional Chinese Medicines Database (TCMD). Molecular simulation indicates that the MOL376 (a compound derived from a Chinese medicine herb with the therapeutic efficacy on the human body such as relieving cough, removing the phlegm, and relieving asthma) has not only the highest binding energy with the receptor but also the good match in geometric conformation. It was observed through docking studies that the van der Waals interactions made substantial contributions to the affinity, and that the receptor active pocket was too large for MDL21870 but more suitable for MOL736. Accordingly, MOL736 might possibly become a promising lead compound for CTSL inhibition for SARS therapy.     

Variation in proton affinity of the guanidino group between free and blocked arginine

Summary. In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), ³¹P NMR, ¹H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.     

The relationship between albumin, other plasma proteins and variables, and age in the acute phase response after liver resection in man

Summary. A large series of plasma albumin (ALB, g/dl) and simultaneous blood and clinical measurements were prospectively performed on 92 liver resection patients, and processed to assess the correlations between ALB, other plasma proteins, additional variables and clinical events. The measurements were performed preoperatively and at postoperative day 1, 3 and 7 in all patients, and subsequently only in those who developed complications or died. In patients who recovered normally ALB was 4.3 ± 0.4 g/dl (mean ± SD) preoperatively, 3.7 ± 0.7 at day 1 and 3, and 3.9 ± 0.4 at day 7. In patients with complications its decrease was more prolonged. In non-survivors it was 3.4 ± 0.4 preoperatively, 3.0 ± 0.4 at day 1, and then decreased further. Regression analysis showed direct correlations between ALB and pseudo-cholinesterase (CHE, U/l, nv 5300-13000), cholesterol (CHOL, mg/dl), iron binding capacity (IBC, mg/dl), prothrombin activity (PA, % of standard reference) and fibrinogen, an inverse correlation with blood urea nitrogen (BUN, mg/dl) for any given creatinine level (CREAT, mg/dl), and weaker direct correlations with hematocrit, other variables and dose of exogenous albumin. An inverse relationship found between ALB and age (AGE, years) became postoperatively (POSTOP) also a function of outcome, showing larger age-related decreases in ALB associated with complications (COMPL: sepsis, liver insufficiency) or death (DEATH). Main overall correlations: CHE = 287.4(2.014)^ALB, r = 0.73; CHOL = 16.5(1.610)^ALB (1.001)^ALKPH, r = 0.71; IBC = 68.6(1.391)^ALB, r = 0.64; PA = 13.8 + 16.0(ALB), r = 0.51; BUN = 21.3 + 20.2(CREAT) – 6.2(ALB), r = 0.91; ALB = 5.0–0.013(AGE) – {0.5 + 0.003(AGE) _COMPL + 0.012(AGE) _DEATH } _POSTOP , r = 0.74 [p < 0.001 for each regression and each coefficient; ALKPH = alkaline phosphatase, U/l, nv 98-279, independent determinant of CHOL; discontinuous variables in italics label the change in regression slope or intercept associated with the corresponding condition]. These results suggest that altered albumin synthesis (or altered synthesis unable to compensate for albumin loss, catabolism or redistribution) is an important determinant of hypoalbuminemia after hepatectomy. The correlations with age and postoperative outcome support the concept that hypoalbuminemia is a marker of pathophysiologic frailty associated with increasing age, and amplified by the challenges of postoperative illness.     

Transport of L-proline,L-proline-containing peptides and related drugs at mammalian epithelial cell membranes

Summary. Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.     

Decrease of plasma taurine in Gaucher disease and its sustained correction during enzyme replacement therapy

Summary. Gaucher disease is caused by an autosomal-recessive deficiency of glucocerebrosidase. Cells of monocytic/macrophagic origin accumulate glucosylceramide. This leads to hepatosplenomegaly, bone destruction, thrombocytopenia and anemia. Enzyme replacement therapy (ERT) with macrophage-targeted glucocerebrosidase leads to normalization of these parameters. The way of macrophage activation in Gaucher disease is not known. Recently, the osmolytes taurine, betaine and inositol were identified as important regulators of macrophage function in liver. Therefore, the role of plasma taurine in Gaucher disease as a primarily macrophage-derived disease was studied. Fasting plasma levels were measured from blood samples of healthy control subjects (n = 29, m : f = 11 : 18, mean age 37 ± 3 years), from un-treated Gaucher patients (n = 16, m : f = 7 : 9, mean age 44 ± 4 years) and those treated for 37 ± 2 months (n = 54, m : f = 19 : 35, mean age 47 ± 2 years). Amino acid analysis was carried out in a BioChrom amino acid analyzer. In the untreated patients, plasma taurine was 45 ± 3 μM, as compared to the controls with a plasma taurine of 63 ± 4 μM (p < 0.01). The aver-age increase of plasma taurine during the first year of ERT was 18 ± 8 μM (n = 10). Patients treated for an average of 37 months (range 1–9 years of ERT) had a plasma taurine of 65 ± 4 μM (n = 54), which was not different from the controls. It is concluded that Gaucher patients show decreased plasma taurine levels and that therapy of Gaucher disease might correct this. It has to be established, whether decreased taurine availability is a cofactor of the permanent activation of glucosylceramide-storing monocytes/macrophages in this disease. Received January 25, 2000/Accepted January 31, 2000