Drosophila egg chamber elongation: Insights into how tissues and organs are shaped

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Insights into in vitro spermatogenesis in mammals: Past,present, future

Considering the self‐renewal and differentiation ability of pluripotent stem cells, some studies have pointed out the possibility of stem cell‐derived sperm production. Most studies that test this hypothesis have been conducted on rodents, with some promising results; however, studies on humans are progressing slowly, and have encountered technical and ethical hurdles. Established methods to differentiate stem cells—including embryoid bodies, co‐culturing, and various feeder cells—may provide a niche that is similar to in vivo conditions and resolve epigenetic abnormalities, but a gonadal‐like three‐dimensional structure is still required to produce germ cells with the correct imprinting. In the last few years, sperm‐like cells with fertilizing capacity were produced from mouse embryonic stem cells, and the resulting embryos from these cells yielded live offspring. Future research should move towards the use of adult stem cells, however, owing to the unavailability of embryonic cells in adults. More intensive research and techniques are required since in vitro spermatogenesis provides hope to individuals without mature sperm who cannot be treated, and may be a useful system to study the precise mechanism of spermatogenesis. In this review, we describe recent studies of in vitro spermatogenesis mechanisms and related techniques in mammals. We also discuss the possible cell surface markers and culture conditions that might improve in vitro spermatogenesis.     

LPA: a novel lipid mediator with diverse biological actions

Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, is a platelet-derived serum factor that evokes a wide range of biological effects, including stimulation of fibroblast proliferation, platelet aggregation, cellular motility, tumour cell invasiveness and neurite retraction. This review summarizes recent insights into the mode of action of LPA. LPA appears to activate its own G-protein-coupled receptor(s) to initiate both classic and novel signal cascades. Of particular interest is LPA's ability to activate the Ras pathway and to stimulate protein tyrosine phosphorylation in concert with remodelling of the actin cytoskeleton.     

Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis

Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in mammalian NATs, possess inherent motions on the nanosecond time scale. The latter segment becomes more restricted in its motions upon substrate binding according to our NMR XNOE data. This greater rigidity appears to stem from interactions with the substrate. Finally, NAT acetylation has been suggested to protect these enzymes from ubiquitination. Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection.     

Getting greasy: how transmembrane polypeptide segments integrate into the lipid bilayer

Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis.     

Insights into a mummy: a paleoradiological analysis

The aim of the study was to analyze possible human skeletal remains within the wrappings of a mummy from the Archaeological Museum, Zagreb, Croatia through the use of the multidetector CT (MDCT) technology. Plain X-ray films and MDCT images of the mummy were taken in both frontal and lateral views. In a single volumetric acquisition of the whole body by MDCT 0.75 mm axial slices were obtained and combined with sagittal and coronal reformatting and three-dimensional (3D) reconstruction. Sex and age was assessed visually using standard anthropological methods. The results suggest that the mummy was of an adult female, most likely over 40 years of age at death. Pathologies observed included degenerative changes on the vertebral column and healed fractures of the lower right arm. Damage of the ethmoid bone at the roof of the nasal cavity was most likely caused by mortuary brain removal practice. Remnants of a resin and an unusual object were found inside the cranial cavity. An elongated metal object and additional three metal "belts" can be seen on the lower portion of the body. All internal organs were removed and thoracic and abdominal cavities were filled with various substances, most likely mud and pieces of linen cloth. Our results show that the MDCT is a very useful technique for assessing the human remains in archeological samples, especially in comparison to the use of plain film (X-ray), where important details are obscured and 3D imaging impossible.     

Insights into lipid raft structure and formation from experiments in model membranes

Rafts are sphingolipid/cholesterol-rich lipid domains believed to exist within certain eukaryotic cell membranes. Model membrane studies have been key to understanding the basic physical principles behind raft formation. Recent fluorescence quenching studies have demonstrated that tight packing between sterols and sphingolipids is the driving force for raft formation, and have begun to decipher the rules governing how different molecules interact with rafts.     

Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.     

Insights into ALS pathomechanisms: from flies to humans

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel “non-obvious” models for the disease.     

Insights into ALS pathomechanisms: from flies to humans

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing the death of motor neurons with consequent muscle atrophy and paralysis. Several neurodegenerative diseases have been modeled in Drosophila and genetic studies on this model organism led to the elucidation of crucial aspects of disease mechanisms. ALS, however, has lagged somewhat behind possibly because of the lack of a suitable genetic model. We were the first to develop a fly model for ALS and over the last few years, we have implemented and used this model for a large scale, unbiased modifier screen. We also report an extensive bioinformatic analysis of the genetic modifiers and we show that most of them are associated in a network of interacting genes controlling known as well as novel cellular processes involved in ALS pathogenesis. A similar analysis for the human homologues of the Drosophila modifiers and the validation of a subset of them in human tissues confirm and expand the significance of the data for the human disease. Finally, we analyze a possible application of the model in the process of therapeutic discovery in ALS and we discuss the importance of novel “non-obvious” models for the disease.     

Insights into archaeal chaperone machinery: a network-based approach

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.