On the Productivity and Properties of l-Asparaginase from Escherichia coli A–l–3

Oxidation of grayanotoxin (GTX) II with lead (IV) acetate in methanol gave a new derivative, the 1(R)-spiro-3,6(S),14,16-tetra-hydroxy-5-keto derivative. Treatment of GTX-II tetraacetate in acetic acid by using Pb(IV) acetate as an oxidizing agent gave a novel 1,5-seco-GTX derivative, Δ¹⁽¹⁰⁾-1,5-seco-GTX-pentaacetate, together with the 1,5-seco-GTX-1(R) derivative. Oxidation of GTX-II-tetraacetate with Tl(III) acetate in acetic acid or benzene gave the 1,5-seco-GTX-1(S) derivative.     

A Revision of the Cytology and Ontogeny of Several Deficiencies in the 3a1–3c6 Region of the X Chromosome of DROSOPHILA MELANOGASTER

The cytology and developmental attributes of 18 deficiency mutations in the 3A1–3C6 region of the salivary gland X chromosome of Drosophila melanogaster have been investigated. The cytological limits of several older deficiencies have been revised and clarified and several new deficiencies are characterized. The deficiency mutants, with one possible exception, show a lethal phase in the late embryonic period or the early first larval instar. In contrast, the earliest acting point mutation lethals exposed by these deficiencies generally exhibit a somewhat later, post-embryonic lethality, perhaps indicating that the deficiencies are having some cumulative or synergistic impact on development. However, even with this difference in time of lethality, it is still possible to conclude that it is not the absolute size of the deficiency but rather the character of the loci deleted that determines the impact on development. Observations on the morphology of lethal embryos shows that while this analysis is internally consistent, it does not agree with earlier work. None of the 3A1–3C6 deficiencies causes any major teratologies during embryogenesis. Furthermore, the "earliest acting" gene in this region does not lie in band 3C1 but is most likely associated with bands 3A8–10.     

A Model of the Hippocampal–cortical Memory System

Based on physiological evidence, we propose a theoretical model of the hippocampal–cortical memory system. The model consists of the following components: the sensory system, the hippocampus (short-term memory), and the association cortex (long-term memory). A series of key codes (local information) is supplied from the sensory system, while context (global information) is inputted from the hippocampus. The two inputs interact dynamically in the association cortex. The interactive neurons work as a detector of coincidence. The cortical network learns the memory information through the coincidence window and, finally, stores it in the form of attractors. This local–global information works as an addressor to designate the stored location of the memory in the association cortex and accelerates the process of storing and retrieving memory information.     

A review of the implication of hypoxanthine excess in the physiopathology of Lesch–Nyhan disease

ABSTRACT

Lesch–Nyhan disease is caused by HGprt deficiency, however, the mechanism by which enzyme deficiency leads to the severe neurological manifestations is still unknown. We hypothesized that hypoxanthine excess leads, directly or indirectly, through its action in adenosine transport, to aberrations in neuronal development. We found that hypoxanthine diminishes adenosine transport and enhances stimulation of adenosine receptors. These effects cause an imbalance between adenosine, dopamine, and serotonin receptors in HGprt deficient cells, and cells differentiated with hypoxanthine showed an increase in dopamine, adenosine and serotonin receptors expression. Hypoxanthine deregulates early neuronal differentiation increasing WNT4 and EN1 gene expression.     

A Study of the Insertion–Deletion Polymorphism of the Serotonin Transporter Gene in Populations from the Volga–Ural Region

The insertion–deletion polymorphism of the serotonin transporter gene (SLC6A4) was studied using the polymerase chain reaction (PCR) in eight populations from the Volga–Ural region (the Bashkir, Chuvash, Tatar, Udmurt, Mari, Mordovian, and Komi populations and the population of Russians living in the Arkhangel'skii raion of Bashkortostan). For this polymorphic system, the pattern of distribution of main population parameters was established in the region studied. Depending on population ethnicity, specific trends were revealed in the pattern of frequencies of alleles and genotypes of geneSLC6A4.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.     

A Computational Exploration of the Interactions of the Green Tea Polyphenol (–)-Epigallocatechin 3-Gallate with Cardiac Muscle Troponin C

Thanks to its polyphenols and phytochemicals, green tea is believed to have a number of health benefits, including protecting from heart disease, but its mechanism of action at the molecular level is still not understood. Here we explore, by means of atomistic simulations, how the most abundant of the green tea polyphenols, (–)-Epigallocatechin 3-Gallate (EGCg), interacts with the structural C terminal domain of cardiac muscle troponin C (cCTnC), a calcium binding protein that plays an important role in heart contractions. We find that EGCg favourably binds to the hydrophobic cleft of cCTnC consistently with solution NMR experiments. It also binds to cCTnC in the presence of the anchoring region of troponin I (cTnI(34–71)) at the interface between the E and H helices. This appears to affect the strength of the interaction between cCTnC and cTnI(34–71) and also counter-acts the effects of the Gly159Asp mutation, related to dilated cardiomyopathy. Our simulations support the picture that EGCg interacting with the C terminal domain of troponin C may help in regulating the calcium signalling either through competitive binding with the anchoring domain of cTnI or by affecting the interaction between cCTnC and cTnI(34–71).     

In Vivo Characterization of the Biodistribution Profile of Amphipol A8–35

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8–35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8–35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10–20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8–35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8–35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.     

A comprehensive resequence analysis of the KLK15–KLK3–KLK2 locus on chromosome 19q13.33

Single nucleotide polymorphisms (SNPs) in the KLK3 gene on chromosome 19q13.33 are associated with serum prostate-specific antigen (PSA) levels. Recent genome wide association studies of prostate cancer have yielded conflicting results for association of the same SNPs with prostate cancer risk. Since the KLK3 gene encodes the PSA protein that forms the basis for a widely used screening test for prostate cancer, it is critical to fully characterize genetic variation in this region and assess its relationship with the risk of prostate cancer. We have conducted a next-generation sequence analysis in 78 individuals of European ancestry to characterize common (minor allele frequency, MAF >1%) genetic variation in a 56 kb region on chromosome 19q13.33 centered on the KLK3 gene (chr19:56,019,829–56,076,043 bps). We identified 555 polymorphic loci in the process including 116 novel SNPs and 182 novel insertion/deletion polymorphisms (indels). Based on tagging analysis, 144 loci are necessary to tag the region at an r ² threshold of 0.8 and MAF of 1% or higher, while 86 loci are required to tag the region at an r ² threshold of 0.8 and MAF >5%. Our sequence data augments coverage by 35 and 78% as compared to variants in dbSNP and HapMap, respectively. We observed six non-synonymous amino acid or frame shift changes in the KLK3 gene and three changes in each of the neighboring genes, KLK15 and KLK2. Our study has generated a detailed map of common genetic variation in the genomic region surrounding the KLK3 gene, which should be useful for fine-mapping the association signal as well as determining the contribution of this locus to prostate cancer risk and/or regulation of PSA expression.     

A nonlinear autoregressive Volterra model of the Hodgkin–Huxley equations

We propose a new variant of Volterra-type model with a nonlinear auto-regressive (NAR) component that is a suitable framework for describing the process of AP generation by the neuron membrane potential, and we apply it to input-output data generated by the Hodgkin–Huxley (H–H) equations. Volterra models use a functional series expansion to describe the input-output relation for most nonlinear dynamic systems, and are applicable to a wide range of physiologic systems. It is difficult, however, to apply the Volterra methodology to the H–H model because is characterized by distinct subthreshold and suprathreshold dynamics. When threshold is crossed, an autonomous action potential (AP) is generated, the output becomes temporarily decoupled from the input, and the standard Volterra model fails. Therefore, in our framework, whenever membrane potential exceeds some threshold, it is taken as a second input to a dual-input Volterra model. This model correctly predicts membrane voltage deflection both within the subthreshold region and during APs. Moreover, the model naturally generates a post-AP afterpotential and refractory period. It is known that the H–H model converges to a limit cycle in response to a constant current injection. This behavior is correctly predicted by the proposed model, while the standard Volterra model is incapable of generating such limit cycle behavior. The inclusion of cross-kernels, which describe the nonlinear interactions between the exogenous and autoregressive inputs, is found to be absolutely necessary. The proposed model is general, non-parametric, and data-derived.     

Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.     

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A–type lamins

Background

The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression.

Methods

In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson–Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses.

Results

Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression.

Conclusions

These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics.

General significance

Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.     

A simple fluid–structure coupling algorithm for the study of the anastomotic mechanics of vascular grafts

Vascular anastomoses constitute a main factor in poor graft performance due to mismatches in distensibility between the host artery and the graft. This work aims at computational fluid–structure investigations of proximal and distal anastomoses of vein grafts and synthetic grafts. Finite element and finite volume models were developed and coupled with a user-defined algorithm. Emphasis was placed on the simplicity of the coupling algorithm. An artery and vein graft showed a larger dilation mismatch than an artery and synthetic graft. The vein graft distended nearly twice as much as the artery while the synthetic graft displayed only approximately half the arterial dilation. For the vein graft, luminal mismatching was aggravated by development of an anastomotic pseudo-stenosis. While this study focused on end-to-end anastomoses as a vehicle for developing the coupling algorithm, it may serve as useful point of departure for further investigations such as other anastomotic configurations, refined modelling of sutures and fully transient behaviour.