Gia Voeltz: shaping ideas about ER shape. Interviewed by Caitlin Sedwick

Gia Voeltz is using in vitro assays, cell biology, and electron microscopy to investigate how the endoplasmic reticulum (ER) acquires its tubular shape and how this shape supports ER function.     

Hydroxylation of dehydroepiandrosterone and its acetate with potato tuber slices

Evidence is presented for the view that the potato tuber tissue contains an unstable enzyme capable of hydroxylation of dehydroepiandrosterone and its acetate in low yield. The products of hydroxylation were isolated and identified. The possibility is discussed that auto-oxidation is a simultaneous reaction.     

New methods allowing the detection of protein aggregates: A case study on trastuzumab

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin®) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.Key words: immunoglobulin, aggregation, stability, protein, trastuzumab, herceptin®, methodology     

Peptide Epitalon activates chromatin at the old age

OBJECTIVES and design. We have studied the effect of synthetic peptide Epitalon on the activity of ribosomal genes, denaturation parameters of total heterochromatin, polymorphism of structural C-heterochromatin and the variability of facultative heterochromatin in cultured lymphocytes of persons aged 76-80 years. RESULTS: The obtained data demonstrate that Epitalon induces the activation of ribosomal genes, decondensation of pericentromeric structural heterochromatin and the release of genes repressed due to the age-related condensation of euchromatic chromosome regions. CONCLUSIONS: Epitalon has shown its ability to activate chromatin by modifying heterochromatin and heterochromatinized chromosome regions in the cells of older persons.     

Cropping Systems and Cultural Practices Determine the Rhizoctonia Anastomosis Groups Associated with Brassica spp. in Vietnam

Ninety seven Rhizoctonia isolates were collected from different Brassica species with typical Rhizoctonia symptoms in different provinces of Vietnam. The isolates were identified using staining of nuclei and sequencing of the rDNA-ITS barcoding gene. The majority of the isolates were multinucleate R. solani and four isolates were binucleate Rhizoctonia belonging to anastomosis groups (AGs) AG-A and a new subgroup of A-F that we introduce here as AG-Fc on the basis of differences in rDNA-ITS sequence. The most prevalent multinucleate AG was AG 1-IA (45.4% of isolates), followed by AG 1-ID (17.5%), AG 1-IB (13.4%), AG 4-HGI (12.4%), AG 2-2 (5.2%), AG 7 (1.0%) and an unknown AG related to AG 1-IA and AG 1-IE that we introduce here as AG 1-IG (1.0%) on the basis of differences in rDNA-ITS sequence. AG 1-IA and AG 1-ID have not been reported before on Brassica spp. Pathogenicity tests revealed that isolates from all AGs, except AG-A, induced symptoms on detached leaves of several cabbage species. In in vitro tests on white cabbage and Chinese cabbage, both hosts were severely infected by AG 1-IB, AG 2-2, AG 4-HGI, AG 1-IG and AG-Fc isolates, while under greenhouse conditions, only AG 4-HGI, AG 2-2 and AG-Fc isolates could cause severe disease symptoms. The occurrence of the different AGs seems to be correlated with the cropping systems and cultural practices in different sampling areas suggesting that agricultural practices determine the AGs associated with Brassica plants in Vietnam.     

Myomesin is a molecular spring with adaptable elasticity

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.     

Rough sheets and smooth tubules

The endoplasmic reticulum (ER) has distinct morphological domains composed of sheets and tubules, which differ in their characteristic membrane curvature. Key proteins may drive the formation of these structural morphologies, which in turn could generate the rough and smooth functional domains of the ER.     

Sequence specificity of psoralen photobinding to DNA: a quantitative approach.

The effects of different DNA sequences on the photoreaction of various furocoumarin derivatives was investigated from a quantitative point of view using a number of self-complementary oligonucleotides. These contained 5'-TA and 5'-AT residues, having various flanking sequences. The furocoumarins included classical bifunctional derivatives, such as 8-methoxy- and 5-methoxypsoralen, as well as monofunctional compounds, such as angelicin and benzopsoralen. Taking into an account the thermodynamic constant for noncovalent binding of each psoralen to each DNA sequence, the rate constants for the photobinding process to each fragment were evaluated. The extent of photoreaction is greatly affected by the DNA sequence examined. While sequences of the type 5'-(GTAC)n are quite reactive towards all furocoumarins, 5'-TATA exhibited a reduced rate of photobinding using monofunctional psoralens. In addition terminal 5'-TA groups were the least reactive with 5- and 8-methoxypsoralen, but not with angelicin or benzopsoralen. Also 5'-AT-containing fragments exhibited remarkably variable responses toward monofunctional or bifunctional psoralen derivatives. As a general trend the photoreactivity rate of the former is less sequence-sensitive, the ratio between maximum and minimum being less than 2 for the examined fragments. The same ratio is about 3.4 for 8-methoxypsoralen and 6.2 for 5-methoxypsoralen. This approach, in combination with footprinting studies, appears to be quite useful for a quantitative investigation of the process of covalent binding of psoralens to specific sites in DNA.