Purification and Some Properties of a Neutral α-Glucosidase from Pig Serum

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s_20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 10⁵ by thin-layer gel filtration and SDS-disc electrophoresis.

The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.     

A New Synthesis of α-Methylspermidine

A five-step synthesis of -methylspermidine (1,8-diamino-5-azanonane), the first polyamine analogue preventing pathological consequences of spermidine depletion in transgenic rats overproducing spermine/spermidine N ¹-acetyltransferase, from ethyl 3-aminobutyrate was achieved in a high overall yield.     

Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (G_αgust) and α-transducin (G_αtran) G protein subunits. This study tested whether G_αtran and G_αgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, G_αgust and G_αtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, G_αgust and G_αtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of G_αtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of G_αgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included G_αtran / 5HT-IR and G_αtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as G_αgust /5-HT-IR or G_αgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in G_αtran-IR cells in the duodenal crypts and a significant increase of G_αgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of G_αtran-G_αgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of G_αtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of G_αgust / G_αtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.     

New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

The causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP⁺, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, α-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology.     

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.     

A New Internal Ribosomal Entry Site 5′ Boundary Is Required for Poliovirus Translation Initiation in a Mouse System

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1–8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.The single-stranded genome of poliovirus has mRNA polarity, is approximately 7,500 nucleotides (nt) in length, is polyadenylylated (45), and is linked covalently at its 5′ end to a small protein called VPg (30, 41). The RNA itself is infectious; cells transfected with the RNA produce progeny virions that are infectious. Poliovirus RNA harbors a long 5′ noncoding region of approximately 740 nt that is important for viral RNA and protein syntheses. A possible cloverleaf-like structure formed by the 5′-proximal end of the RNA (approximately 90 nt) is a probable cis element that regulates the synthesis of the plus-strand RNA (1). nt 120 to 590 of the poliovirus RNA make up the internal ribosomal entry site (IRES) (32), which directs the viral translation initiation step in a 5′-end- and cap-independent manner (17, 25, 29, 44). The IRES is assumed to carry a number of secondary structures (10, 40), and multiple host cellular factors are required for its functions.Translation of poliovirus does not occur in a cell-free wheat germ translation system, and it occurs only inefficiently and usually incorrectly in rabbit reticulocyte lysates (RRL) (9). The poor translation in RRL, however, is markedly improved by the addition of factors from HeLa cells (5, 9, 33). Other IRESs, such as the IRESs of encephalomyocarditis virus RNA (18) and hepatitis C virus RNA (43), are highly functional in the RRL system. These observations indicate that individual IRESs with different structures may require quantitatively and/or qualitatively different sets of host factors for their activities.Determinants for strain-specific neurovirulence (replication ability) of poliovirus type 1 in the central nervous system (CNS) have been mapped in the IRES region, particularly at nt 480 of the genome, by using monkey neurovirulence tests on recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains (19, 24). Similar results were obtained when the recombinants were tested for their relative neurovirulence levels by using transgenic (Tg) mice carrying the human gene for the poliovirus receptor (15, 20, 34). Thus, the IRES seems to be an important regulatory element for strain-specific expression of poliovirus neurovirulence. These two animal models show no difference in the development of the disease, even though replication of the virus in vivo must involve a number of biological interactions between viral and host factors. These results suggest that host factors of monkeys and mice, including IRES-related factors, support the expression of poliovirus neurovirulence (replication) in much the same way. However, it is possible that species differences between the IRES-related host factors of monkeys and mice exist.Several mutants with alterations in the stem-loop II (SLII) region were constructed from an infectious cDNA clone of the virulent Mahoney strain of poliovirus type 1 (39). The mutants replicated well in primate cells and in the CNS of monkeys but did poorly in mouse cells expressing human poliovirus receptor and in the CNS of the Tg mice carrying the human PVR gene (39). The replication of the mutant strains in mouse cells was blocked at the IRES-dependent translation initiation step, indicating that the function of the SLII as a part of the IRES is deficient in mouse cells but still active in primate cells. These differences in how the SLII mutants acted in the two animal models point to an interaction between the SLII and SLII-related host factors that could be a determinant for host-specific replication of poliovirus.To gain a deeper insight into the molecular basis of the function of the SLII region within the IRES, revertants that acquired the ability to replicate in mouse cells were isolated from the SLII mutants. Genetic analysis of mutation sites in the revertant genomes revealed that nt 107 within the 5′ noncoding region of poliovirus RNA influenced the efficiency of the IRES-dependent translation initiation process and that the remaining mutation sites (nt 120 to 161), in addition to nt 107, were required for the expression of the full revertant phenotype.     

Isolation of α2 Macroglobulin Subunits from Human Serum

Subunits of α₂ macroglobulin (α₂M) with sedimentation rates lower than 10 S were found in ultrafiltrates (through macroglobulin-stopping membranes) of normal human sera. Five to six percent of the total α₂M in human sera exists in subunit form. After isolation by means of ammonium sulfate precipitation, curtain electrophoresis, gel filtration and ultrafiltration these subunits were shown to have sedimentation rates of 5–10 S.     

Transport of α-Ketoisocaproate in Neuroblastoma NB-2a Cells

Transport of α-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free ketoacid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and β-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, α-cyano-4-hydroxycinnamate, 4,4′-diisothiocyano-2,2′-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na⁺].     

A Transmembrane Polar Interaction Is Involved in the Functional Regulation of Integrin αLβ2

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of α and β subunits. Each subunit contains a single α-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of αβ TM packing. The leukocyte integrin αLβ2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of αLβ2 TMs is consistent with that of the integrin αIIbβ3 TMs. However, molecular dynamics simulations of αLβ2 TMs in lipids predicted a polar interaction involving the side chains of αL Ser1071 and β2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled αLβ2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of αLβ2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of β2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated αLβ2, αMβ2, and αXβ2 in 293T transfectants. We also show that the expression of mutant β2 Thr686Gly in β2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1α treatment as compared to wild-type β2-expressing cells. These two TM polar residues are totally conserved in other members of the β2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of αLβ2 function.     

Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend”

Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α₁-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was “fished out” (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.     

A New Flow-Regulating Cell Type in the Demosponge Tethya wilhelma – Functional Cellular Anatomy of a Leuconoid Canal System

Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system, flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma, which we studied using scanning electron microscopy. We found a hitherto undescribed cell type, the reticuloapopylocyte, which is involved in flow regulation in the choanocyte chambers. It has a highly fenestrated, grid-like morphology and covers the apopylar opening. The minute opening of the reticuloapopylocyte occurs in an opened, intermediate and closed state. These states permit a gradual regulation of the total apopylar opening area. In this paper the three states are included in a theoretical study into flow conditions which aims to draw a link between functional cellular anatomy, the hydrodynamic situation and the regular body contractions seen in T. wilhelma. This provides a basis for new hypotheses regarding the function of bypass elements and the role of hydrostatic pressure in body contractions. Our study provides insights into the local and global flow conditions in the sponge canal system and thus enhances current understanding of related physiological processes.     

Serum Antibodies against CD28– A New Potential Marker of Dismal Prognosis in Melanoma Patients

Background

Autoantibodies against CD28 have been found in patients with autoimmune and atopic diseases. These antibodies may act as superagonists and activate T cells but may also be antagonistic or induce immunosuppressive effects by activating regulatory T cells. Autoimmunity in melanoma patients has been discussed controversially.

Objective

We investigated 230 melanoma patients for the occurrence of CD28 antibodies and the effect of the latter on overall and progress-free survival.

Methods

We constructed an ELISA assay to measure CD28 serum antibodies. 230 patients with melanoma and a control-group of 625 patients consistent of 212 patients with virus hepatitis b or c, 149 patients with allergies, 78 patients with psoriasis, 46 patients with plasmocytoma and 140 healthy blood donors were investigated for the occurrence of CD28 antibodies.

Results

CD28 abs occur at a higher percentage in patients with melanoma and in patients with viral hepatitis than in other groups investigated (p<0.001). Occurrence of CD28 abs is significantly higher in patients receiving interferons independent from the underlying disease (p<0.001). In vitro CD28 serum antibodies have an inhibitory effect on the CD28 receptor as they lead to reduced stimulation of Jurkat cells. Presence of CD28 was correlated with a higher risk of dying from melanoma (p = 0.043), but not with a significantly shortened overall survival or progression-free survival.

Conclusion

Interferon therapy appears to induce the production of CD28 abs. In light of reports that these CD28 abs induce immunosuppressive Tregs and – as our data show – that they are inhibitors of CD28 receptor mediated stimulation, the continuation of therapies with interferons in melanoma patients developing CD28 antibodies should be critically reconsidered, since our data indicate a worse outcome of patients with CD28 abs.