Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of -amylase mRNA in the aleurone layer. In addition, cell wall degradation started in cells near the embryo on the sides facing the endosperm. Subsequently cell wall degradation extended to the lateral cell walls and to cells more to the distal end of the grain. A typical alteration of the nucleus was observed by electron microscopy and an almost complete degradation of DNA was found in the nucleus while the nuclear envelope remained intact. The results indicate that programmed cell death occurred in aleurone cells during germination. A model is proposed for the regulation of programmed cell death in aleurone cells during germination involving ABA levels and cell wall degradation.     

Methyl palmitate does not elicit invasion of honeybee brood cells by Varroa mites

Since Varroa mites decide at some distance from the larva whether to stay on a bee or invade a cell, they may well use a volatile chemical to select a brood cell. A few aliphatic esters, predominantly methyl palmitate, have been claimed to be this volatile signal for the mites for two reasons. The mites respond to the esters in an olfactometer (Le Conte et al., 1989), and the levels of the esters in worker and drone larvae may explain that drone cells are attractive during a longer period and are invaded more frequently than worker cells (Trouiller et al., 1992). However, invasion itsclf appeared to be unaffected by application of methyl palmitate to brood cells. In addition, analysis of the volatiles emanating from attractive brood cells showed hundreds of components in the volatile blend, but in only 2 of 17 analyses a trace of methyl palmitate was found. Hence, there is no reason to believe that methyl palmitate is used as a signal for invasion by the mites.     

A single histidine residue modulates enzymatic activity in acidic mammalian chitinase

Mammals express two active chitinases, chitotriosidase and AMCase. Only AMCase displays an extremely acidic pH optimum, consistent with its observed presence in the gastro-intestinal tract. A structural model of AMCase reveals the presence of a conserved histidine residue in the active site. Mutational analyses and molecular dynamics simulations show that His187 is responsible for the acidic optimum and suggest pH dependent modulation of the reaction mechanism that is unique to AMCases. Concluding, His187 is a crucial structural component of the active site of AMCase and this unique feature may serve as a lead for the development of specific inhibitors.     

Long-term effects of growth hormone therapy on bone mineral density,body composition,and serum lipid levels in growth hormone deficient children: a 6-year follow-up study

AIM: To study the effects of growth hormone (GH) deficiency (GHD) and GH replacement therapy (GHRx) on bone mineral density (BMD) and body composition. METHODS: 59 GHD children participated (age range 0.4-16.9 years); the follow-up period was 6 years. Lumbar spine BMD (BMD(LS)), total-body BMD (BMD(TB)), and body composition were measured prospectively using dual-energy X-ray absorptiometry. RESULTS: Mean BMD(LS )and BMD(TB) were significantly reduced at the time of the diagnosis. The bone mineral apparent density of the lumbar spine (BMAD(LS)) was reduced to a lesser degree. The BMAD(LS) increased to normal values after 1 year; BMD(LS) and BMD(TB) normalized 1 year later. At the time of the diagnosis, the lean body mass was reduced and steadily increased during GHRx. Percentage of body fat was increased at baseline and normalized within 6 months. The severity of GHD was not associated with the BMD at diagnosis or the response to GHRx. CONCLUSION: Areal BMD(LS) and BMD(TB) and, to a lesser extent, BMAD(LS) are decreased in GHD children, but normalize within 1-2 years.     

Exchange of the C-terminal part of VP3 from very virulent infectious bursal disease virus results in an attenuated virus with a unique antigenic structure

Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).     

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.     

Biochemistry of glycosphingolipid storage disorders: implications for therapeutic intervention

The physiological importance of the degradative processes in lysosomes is revealed by the existence of at least 40 distinct inherited diseases, the so-called lysosomal storage disorders. Most of these diseases are caused by a deficiency in a single lysosomal enzyme, or essential cofactor, and result in the lysosomal accumulation of one, or sometimes several, natural compounds. The most prevalent subgroup of the lysosomal storage disorders is formed by the sphingolipidoses, inherited disorders that are characterized by excessive accumulation of one or multiple (glyco)sphingolipids. The biology of glycosphingolipids has been extensively discussed in other contributions during this symposium. This review will therefore focus in depth on (type 1) Gaucher disease, a prototypical glycosphingolipidosis. The elucidation of the primary genetic defect, being a deficiency in the lysosomal glucocerebrosidase, is described. Characterization of glucocerebrosidase at protein and gene level has subsequently opened avenues for therapeutic intervention. The development of successful enzyme replacement therapy for type 1 Gaucher disease is discussed. Attention is also paid to the alternative approach of substrate modulation using orally administered inhibitors of glucosylceramide synthesis. Novel developments about the monitoring of age of onset, progression and correction of disease are described. The remaining challenges about pathophysiology of glycosphingolipidoses are discussed in view of further improvements in therapy for these debilitating disorders.     

Modifications of the 3'-UTR stem-loop of infectious bursal disease virus are allowed without influencing replication or virulence

Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3′-untranslated regions (UTRs) of the coding strand of both genomic segments are able to fold into a single stem–loop structure. To assess the determinants for a functional 3′-UTR, we mutagenized the 3′-UTR stem–loop structure of the B-segment. Rescue of infectious virus from mutagenized cDNA plasmids was impaired in all cases. However, after one passage, the replication kinetics of these viruses were restored. Sequence analysis revealed that additional mutations had been acquired in most of the stem–loop structures, which compensated the introduced ones. A rescued virus with a modified stem–loop structure containing four nucleotide substitutions, but preserving its overall secondary structure, was phenotypically indistinguishable from wild-type virus, both in vitro (cell culture) and in vivo (chickens, natural host). Sequence analysis showed that the modified stem–loop structure of this virus was fully preserved after four serial passages. Apparently, it is the stem–loop structure and not the primary sequence that is the functional determinant in the 3′-UTRs of IBDV.     

Leaf traits and herbivory rates of tropical tree species differing in successional status

We evaluated leaf characteristics and herbivory intensities for saplings of fifteen tropical tree species differing in their successional position. Eight leaf traits were selected, related to the costs of leaf display (specific leaf area [SLA], water content), photosynthesis (N and P concentration per unit mass), and herbivory defence (lignin concentration, C:N ratio). We hypothesised that species traits are shaped by variation in abiotic and biotic (herbivory) selection pressures along the successional gradient. All leaf traits varied with the successional position of the species. The SLA, water content and nutrient concentration decreased, and lignin concentration increased with the successional position. Herbivory damage (defined as the percentage of damage found at one moment in time) varied from 0.9-8.5% among the species, but was not related to their successional position. Herbivory damage appeared to be a poor estimator of the herbivory rate experienced by species, due to the confounding effect of leaf lifespan. Herbivory rate (defined as percentage leaf area removal per unit time) declined with the successional position of the species. Herbivory rate was only positively correlated to water content, and negatively correlated to lignin concentration, suggesting that herbivores select leaves based upon their digestibility rather than upon their nutritive value. Surprisingly, most species traits change linearly with succession, while resource availability (light, nutrients) declines exponentially with succession.     

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.