A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function

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Effect of the leader sequence on the expression of recombinant C. rugosa lipase by S. cerevisiae cells

Summary The Candida rugosa lipase I gene has been expressed in Saccharomyces cerevisiae. The recombinant lipase was efficiently synthesized only following the replacement of the enzyme endogenous leader sequence with the signal peptide of the Kluyveromyces lactis killer toxin. Amount of accumulated lipase was about 10–20 mg/l in batch culture and over 1g/l in a computer-controlled fed-batch fermentation system.     

A Soap Technique for Cell Separation to Study the Seed Coat of Sesbania punicea

A technique is described for separating plant cells used for morphological studies. The plant material is placed in a concentrated solution of olive oil castile soap for 1-2 days or more. The material is then thoroughly washed and placed between two glass slides. The upper glass slide is lifted from the lower one, then gently pressed down several times. Through this procedure Malpighian cells of the seed coat of Sesbania punicea. mesophyll cells of Euphorbia peplus and of Trifolium pratense and cortical cells of the aerial roots of Monstera deliciosa have been separated. Various shapes of the Malpighian cells of the Sesbania punicea seed coat can be observed along with intermediates.     

Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii

LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the core of Photosystem II (PSII) to form multiprotein complexes. Several PSII supercomplexes with different antenna sizes have been purified, the largest of which contains three LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13 Å resolution was obtained allowing the reconstruction of the 3D structure of the supercomplex. The position and orientation of the S trimer are the same as in plants; trimer M is rotated by 45° and the additional trimer (named here as LHCII-N), which is taking the position occupied in plants by CP24, is directly associated with the core. The analysis of supercomplexes with different antenna sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the trimers in the PSII supercomplex, while LhcbM5 is part of the “extra” LHCII pool not directly associated with the supercomplex. It is also shown that Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex from plants and a blue shifted absorption spectrum. Finally the data indicate that there are at least six LHCII trimers per dimeric core in the thylakoid membranes, meaning that the antenna size of PSII of C. reinhardtii is larger than that of plants.     

Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals

Background

The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.

Methods and Findings

We immortalized IgG⁺ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.

Conclusions

This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.     

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.     

Pathophysiology and therapy for haemoglobinopathies. Part II: thalassaemias

Thalassaemias result from mutations of the globin genes that cause reduced or absent haemoglobin production and thus interfere with the critical function of oxygen delivery. They represent the most common single-gene disorders, with 4.83% of the world population carrying globin gene variants. Reduced or absent alpha-globin (alpha-thalassaemia) or beta-globin (beta-thalassaemia) leads to anaemia and multifaceted clinical syndromes. In this second of two reviews on the pathophysiology of haemoglobinopathies, we describe the clinical features, pathophysiology and molecular basis of alpha- and beta-thalassaemias. We then discuss current targeted therapies, including the new oral iron chelators, which, along with chronic transfusions, constitute the mainstay of symptomatic therapy for the majority of patients. Finally, we describe potentially curative therapies, such as bone marrow transplant, and discuss some of the outstanding research studies and questions, including the upcoming field of gene therapy for beta-thalassaemia. An accompanying article on haemoglobinopathies (Part I) focuses on sickle cell disease.