Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Substrate reduction therapy in mouse models of the glycosphingolipidoses

Substrate reduction therapy uses small molecules to slow the rate of glycolipid biosynthesis. One of these drugs, N-butyldeoxynojirimycin (NB-DNJ), shows efficacy in mouse models of Tay-Sachs, Sandhoff and Fabry diseases. This offers the prospect that NB-DNJ may be of therapeutic benefit, at least in the juvenile and adult onset variants of these disorders. The infantile onset variants will require an additional enzyme-augmenting modality if the pathology is to be significantly improved. A second drug, N-butyldeoxyglactonojirimycin, looks very promising for treating storage diseases with neurological involvement as high systemic dosing is achievable without any side-effects.     

Chemical Characterization of Gallstones: An Approach to Explore the Aetiopathogenesis of Gallstone Disease in Sri Lanka

Introduction

Records on gallstones and associated ailments in Sri Lankan community are scarce, despite frequent detection of gallstone disease. Identification of the chemical composition of gallstones in the local setting is important in defining aetiopathogenic factors which in turn are useful in implementing therapeutic and preventive strategies. This study aimed to describe the chemical composition of gallstones and the socio-demographic factors of a cohort of Sri Lankan patients with gallstone disease.

Materials and Methods

Data on clinical and socio-demographic factors, and gallstones removed at surgery were collected from patients with cholelithiasis admitted to Teaching Hospital, Peradeniya, Sri Lanka from May 2011 to December 2012. External and cross sectional morphological features of gallstones were recorded by naked eye observation. Compositional analysis was carried out by Fourier Transform Infrared Spectroscopy, X - ray Powder Diffraction, and Atomic Absorption Spectrophotometry. Scanning Electron Microscopy was used to identify the microstructure of gallstones.

Results

Data of 102 patients were analyzed. Of them majority (n = 77, 76%) were females with a female: male ratio of 3:1. Mean age of the study group was 46.1±11.6 years. All the patients had primary gallbladder stones. According to the physical and chemical analysis, majority (n = 54, 53%) were pigment gallstones followed by mixed cholesterol gallstones (n = 38, 37%). Only 10 (9%) had pure cholesterol gallstones. Calcium bilirubinate, calcium carbonate and calcium phosphate were the commonest calcium salts identified in pigment gallstones and core of mixed cholesterol gallstones.

Conclusion

Presence of a pigment nidus in gallstones is a common feature in majority of Sri Lankan patients denoting the possible role of elevated unconjugated bilirubin in bile on the pathogenesis of GS. Hence it is imperative to explore this further to understand the aetiopathogenesis of GS among Sri Lankans.     

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.     

R5-SHIV induces multiple defects in T cell function during early infection of rhesus macaques including accumulation of T reg cells in lymph nodes

Background

HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues.

Methods

Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation.

Results/Conclusions

We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection.     

Storage solutions: treating lysosomal disorders of the brain

Many neurodegenerative diseases are characterized by the accumulation of undegradable molecules in cells or at extracellular sites in the brain. One such family of diseases is the lysosomal storage disorders, which result from defects in various aspects of lysosomal function. Until recently, there was little prospect of treating storage diseases involving the CNS. However, recent progress has been made in understanding these conditions and in translating the findings into experimental therapies. We review the developments in this field and discuss the similarities in pathological features between these diseases and some more common neurodegenerative disorders.     

Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.

Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.     

Subcellular distribution and relative expression of fibrocyte markers in the CD/1 mouse cochlea assessed by semiquantitative immunogold electron microscopy

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.