Evidence for the presence of β-subunit of hexosaminidase in a case of Sandhoff disease using a blotting technique

Hexosaminidases, lysosomal enzymes whose deficiency is responsible for several genetic disorders, exist as two major forms: form A, containing two types of subunits alpha and beta; and form B, containing only beta subunits. We have used a technique involving successively electrophoresis of denatured proteins, transfer (blotting) onto nitrocellulose, and labelling by appropriate antibodies raised against the dissociated forms of hexosaminidases A and B. This technique allows the detection of alpha and beta subunits in crude extracts of normal tissues. The presence of beta chains was demonstrated in the liver of a fetus affected with Sandhoff's disease, deficient in functional hexosaminidases A and B.     

Global phosphorylation at Ser16 of the 32-residue cytoplasmic domain of phospholamban: Comparison of di-t-butyl- and dibenzyl-N,N-diisopropylphosphoramidites

Summary Phospholamban (PLB), a 52-residue integral membrane protein of the cardiac sarcoplasmic reticulum, is known as the regulator of the Ca²⁺-ATPase pump via its phosphorylation-dephosphorylation at Ser¹⁶. In order to investigate the structural effects of the phosphorylation on the cytoplasmic domain of PLB, we synthesized PLB 2–33 in its nonphosphorylated and phosphorylated forms using Fmoc chemistry. PLB 2–33 was phosphorylated using the global phosphorylation method. Phosphorylation with di-t-butyl-N,N-diisopropylphosphoramidite led to an incomplete reaction (nonphosphorylated peptide was recovered) and to the formation of an H-phosphonate. In contrast, the use of dibenzyl-N,N-diisopropylphosphoramidite yielded the desired phosphorylated peptide quantitatively and did not give rise to the H-phosphonate. Our results show the effectiveness of the dibenzyl-protected phosphoramidite when the site to be phosphorylated is particularly hindered.     

Short Communication: Urinary excretion of glutamine transaminase K as an early index of mercuric chloride-induced nephrotoxicity

The possibili that urinary glutamine transaminase K activity might be a marker of a proximal tubule segment-specific response to mercuric chloride was investigated in male rats after a single i.p. injection in time-course and dose-response experiments. Urinary total proteins and angiotensin converting enzyme activity were determined simultaneously. Urinary indices showed an early increase (within 5 h of treatment) of total proteins and angiotensin converting enzyme, whereas glubmine transaminase K increased 10 h after treatment. The peak of all these indices was observed 24 h after mercuric chloride injection. The lowest dose that induced a significant increase in proteins and enzymes was 0.25 mg kg^-1; in addition, a dose-response effect was observed. Glutamine transaminase K appeared to be an early and sensitive index of response of mercuric chloride effects, similar to total proteins and angiotensin converting enzyme. It is suggested that this enzyme is mainly localized in the 'pars recta' of the proximal tubule. Therefore glutamine transaminase K might be a segment-specific marker for the detection of damage localized in this portion of the proximal tubule.     

Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h⁻¹ – Relative Fluorescence Unit h⁻¹), in comparison to batch cultivation (174.0 RFU h⁻¹) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L⁻¹.     

Assessing environmental requirements effects on forest fragmentation sensitivity in two arboreal rodents

The study of the effect of habitat fragmentation on species that inhabit residual patches requires the investigation of the relationship existing between species distribution and landscape components. To understand which components of landscape mosaics are more influential for species’ persistence, we compared the distribution of two arboreal rodents proved to be sensitive to habitat fragmentation, the hazel dormouse Muscardinus avellanarius and the red squirrel Sciurus vulgaris. Their occurrence in residual oak woods in central Italy was studied with nest-boxes and hair-tubes, developing a new method for hair analysis. Their distributions were analysed considering patch, matrix composition and configuration, and landscape vegetation variables. The two species showed a different degree of plasticity, with the squirrel being significantly more specialised at the landscape scale. The comparison of the two distribution patterns highlighted the influence of different ecological constraints and the existence of different strategies to cope with fragmentation. Patch size and patch attributes were generally weaker determinants of occurrence, compared to landscape metrics. The squirrel presence was significantly influenced by the presence of shared perimeter between hedgerows and woods and by the lack of isolation of the residual patches, suggesting the use of several fragments to compensate the low habitat quality. Conversely the hazel dormouse seemed to be more affected by the internal management of the woods, and in particular by the mean DBH. Our results highlight how the recognition of the extrinsic constraints and the influence of multi-scale habitat selection may help guiding land use management, to ensure species conservation in profoundly exploited landscapes.     

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the α-galactosidase A gene and detection of carriers in Fabry disease

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range. Received: 9 April 1996 / Revised: 8 July 1996