Complex I and energy thresholds in the brain

Mitochondrial electron transport chain (ETC) deficiencies are thought to underlie defects in energy metabolism and have been implicated in the neurodegenerative process. In particular, reductions in complex I activities in Parkinson's disease are thought to cause bioenergetic dysfunction with subsequent degeneration of dopaminergic neurons. In terms of bioenergetics and assessing ETC-related problems in the brain, the presence of heterogeneous mitochondria has complicated matters as isolated non-synaptic mitochondria have different energy thresholds and flux control coefficients compared to isolated mitochondria of synaptic origin. The molecular mechanisms that underlie complex I deficiencies in the parkinsonian brain are unknown and are the source of intensive research. This review explores the relationship between complex I activity and energy metabolism in the brain as well as the nature of the complex I defect.     

All-trans retinoic acid prevents development of cardiac remodeling in aortic banded rats by inhibiting the renin-angiotensin system

This study was designed to determine the effect of all-trans retinoic acid (RA) on the development of cardiac remodeling in a pressure overload rat model. Male Sprague-Dawley rats were subjected to sham operation and the aortic constriction procedure. A subgroup of sham control and aortic constricted rats were treated with RA for 5 mo after surgery. Pressure-overloaded rats showed significantly increased interstitial and perivascular fibrosis, heart weight-to-body weight ratio, and gene expression of atrial natriuretic peptide and brain natriuretic peptide. Echocardiographic analysis showed that pressure overload induced systolic and diastolic dysfunction, as evidenced by decreased fractional shortening, ejection fraction, stroke volume, and increased E-to-E(a) ratio and isovolumic relaxation time. RA treatment prevented the above changes in cardiac structure and function and hypertrophic gene expression in pressure-overloaded rats. RA restored the ratio of Bcl-2 to Bax, inhibited cleavage of caspase-3 and -9, and prevented the decreases in the levels of SOD-1 and SOD-2. Pressure overload-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by RA, via upregulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2. The pressure overload-induced production of angiotensin II was inhibited by RA via upregulation of expression of angiotensin-converting enzyme (ACE)2 and through inhibition of the expression of cardiac and renal renin, angiotensinogen, ACE, and angiotensin type 1 receptor. Similar results were observed in cultured neonatal cardiomyocytes in response to static stretch. These results demonstrate that RA has a significant inhibitory effect on pressure overload-induced cardiac remodeling, through inhibition of the expression of renin-angiotensin system components.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.     

Genomic and proteomic perspectives in cell culture engineering.

In the last few years, the number of biologics produced by mammalian cells have been steadily increasing. The advances in cell culture engineering science have contributed significantly to this increase. A common path of product and process development has emerged in the last decade and the host cell lines frequently used have converged to only a few. Selection of cell clones, their adaptation to a desired growth environment, and improving their productivity has been key to developing a new process. However, the fundamental understanding of changes during the selection and adaptation process is still lacking. Some cells may undergo irreversible alteration at the genome level, some may exhibit changes in their gene expression pattern, while others may incur neither genetic reconstruction nor gene expression changes, but only modulation of various fluxes by changing nutrient/metabolite concentrations and enzyme activities. It is likely that the selection of cell clones and their adaptation to various culture conditions may involve alterations not only in cellular machinery directly related to the selected marker or adapted behavior, but also those which may or may not be essential for selection or adaptation. The genomic and proteomic research tools enable one to globally survey the alterations at mRNA and protein levels and to unveil their regulation. Undoubtedly, a better understanding of these cellular processes at the molecular level will lead to a better strategy for 'designing' producing cells. Herein the genomic and proteomic tools are briefly reviewed and their impact on cell culture engineering is discussed.     

Human cell lines for biopharmaceutical manufacturing: history,status, and future perspectives

Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Eriocaulon gopalakrishnanum sp. nov. (Eriocaulaceae) from the Western Ghats,India

Eriocaulon gopalakrishnanum K. Rashmi & G. Krishnakumar sp. nov. is described and illustrated from the low altitude monsoon vegetation in the coastal lateritic plateau of the Western Ghat region of Kerala, India. The new species is distinct in the leaves and spathe being papillose, a character not displayed by any other species from this region.     

Biology of <Emphasis Type="Italic">B. sorokiniana</Emphasis> (syn. <Emphasis Type="Italic">Cochliobolus sativus</Emphasis>) in genomics era

Bipolaris sorokiniana (Sacc.) Shoemaker is a hemi-biotrophic fungal pathogen, which is an anamorph (teleomorph Cochlibolus sativus). It causes spot blotch, root rot and leaf spot diseases in a number of cereals including wheat, barley and other small grain cereals. In the genomics era, the fungus has been subjected to a variety of studies using molecular approaches. Correct chromosome number was determined and molecular karyotypes were prepared using contour-clamped homogeneous electric field. Molecular maps were prepared using markers like RFLPs, SSRs, RAPDs and SNPs. For this purpose, segregating progenies derived from crosses between diverse isolates of the pathogen were used. Whole genome sequencing (WGS) data was collected not only for B. sorokiniana isolates, but also for several species of Cochliobolus. Genes involved in secondary metabolism and virulence were identified from genome sequences. The WGS data has also been utilized for comparative genomics giving useful information about evolutionary trends. A brief account of this information is presented in this review.     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.