The effect of cholesterol in a liposomal Muc1 vaccine

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.     

Biochemical and immunological changes on oral glutamate feeding in male albino rats

 High altitude stress leads to lipid peroxidation and free radical formation which results in cell membrane damage in organs and tissues, and associated mountain diseases. This paper discusses the changes in biochemical parameters and antibody response on feeding glutamate to male albino Sprague Dawley rats under hypoxic stress. Exposure of rats to simulated hypoxia at 7576 m, for 6 h daily for 5 consecutive days, in an animal decompression chamber at 32±2° C resulted in an increase in plasma malondialdehyde level with a concomitant decrease in blood glutathione (reduced) level. Supplementation of glutamate orally at an optimal dose (27 mg/kg body weight) in male albino rats under hypoxia enhanced glutathione level and decreased malondialdehyde concentration significantly. Glutamate feeding improved total plasma protein and glucose levels under hypoxia. The activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) and the urea level remained elevated on glutamate supplementation under hypoxia. Glutamate supplementation increased the humoral response against sheep red blood cells (antibody titre). These results indicate a possible utility of glutamate in the amelioration of hypoxia-induced oxidative stress. Received: 23 March 1998 / Accepted: 19 October 1998     

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes

Myelin-associated glycoprotein (MAG) has been implicated in inhibition of nerve regeneration in the CNS. This results from interactions between MAG and the Nogo receptor and gangliosides on the apposing axon, which generates intracellular inhibitory signals in the neuron. However, because myelin-axon signaling is bidirectional, we undertook an analysis of potential MAG-activated signaling in oligodendrocytes (OLs). In this study, we show that antibody cross-linking of MAG on the surface of OLs (to mimic axonal binding) leads to the redistribution of MAG into detergent (TX-100)-insoluble complexes, hyperphosphorylation of Fyn, dephosphorylation of serine and threonine residues in specific proteins, including lactate dehydrogenase and the beta subunit of the trimeric G-protein-complex, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. We propose that these changes are part of a signaling cascade in OLs associated with MAG function as a mediator of axon-glial communication which might have implications for the mutual regulation of the formation and stability of axons and myelin.     

Qualification of analytical instruments for use in the pharmaceutical industry: A scientific approach

The purpose of the use of analytical instruments is to generate reliable data. Instrument qualification helps fulfill this purpose. No authoritative guide exists that considers the risk of instrument failure and combines that risk with users' scientific knowledge and ability to use the instrument to deliver reliable and consistent data. In the absence of such a guide, the qualification of analytical instruments has become a subjective and often fruitless document-generating exercise. Taking its cue from the new FDA initiative, “Pharmaceutical GMP's for the 21st Century,” an efficient, science- and risk-based process for AIQ was discussed at a workshop on analytical instrument qualification. This report represents the distillate of deliberations on the complicated issues associated with the various stages of analytical instrument qualification. It emphasizes AIQ's place in the overall process of obtaining quality reliable data from analytical instruments and offers an efficient process for its performance, one that focuses on scientific value rather than on producing documents. Implementing such a process should remove ambiguous interpretations by various groups.     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

Studies of apoptosis and bcl-2 in experimental atherosclerosis in rabbit and influence of selenium supplementation

Apoptosis is a ubiquitous physiological mechanism of cell death regulating tissue mass and architecture. An attempt was made in the present study to see the occurrence of apoptotic cell death in three different treatment groups of rabbits viz. Control, HFD fed and HFD + Selenium fed. Apoptotic activity as checked by in situ DNA end labelling (TUNEL Assay) revealed excessive staining, mostly concentrated in plaque region both in fibrous as well as atheromatous plaque in HFD fed animals. However, in selenium (Se) supplemented animals, very little TUNEL staining could be seen, and even that confined to endothelial cells only. The control group on the other hand was totally devoid of any staining. Transmission Electron Microscopic (TEM) study also depicted the occurrence of apoptosis in aortic cells of HFD fed animals and very little in Se supplemented animals. Apoptotic activity has been discussed in relation to oxidative stress in HFD fed group. bcl-2, though an antiapoptotic oncoprotein, was found to be expressed more in atherogenic group as compared to control/HFD + Se treatment. On the whole, the study highlighted the occurrence of apoptotic process in atherosclerosis and the role of Se, a potent antioxidant, in inhibition of apoptotic process in HFD fed animals.     

Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.     

Antifertility,antibacterial, antifungal and percent disease incidence aspects of macrocyclic complexes of manganese(II)

Macrocyclic complexes of Mn(II) were synthesized by template condensation using 2,6-diaminopyridine and diethylenetriamine with malonic, succinic, glutaric and adipic acids. The reaction proceeded smoothly to completion. These 16- to 24-membered N(6), but behaving as tetradentate, macrocyclic complexes were characterized by elemental analyses, molecular weight determinations, infrared, electronic, mass and X-ray spectral analyses. The elemental analyses are consistent with the formation of complexes [Mn(N(6)L(n))Cl(2)]. All the complexes are stable and monomeric in nature, as indicated by the molecular weight determinations. The spectral studies confirmed the proposed framework of the new macrocyclic complexes and indicated an octahedral geometry around the central metal atom. The complexes were screened in vitro against a number of pathogenic fungi and bacteria to assess their growth-inhibiting potential. The testicular sperm density, sperm morphology, sperm motility, density of cauda epididymis, spermatozoa and fertility in mating trials and the biochemical parameters of the reproductive organs of the rat were examined and are discussed.     

Metastatic sweat gland adenocarcinoma: A clinico-pathological dilemma

Background  

Sweat gland adenocarcinoma is a rare malignancy with high metastatic potential seen more commonly in later years of life. Scalp is the most common site of occurrence and it usually spreads to lymph nodes. Liver, lung and bones are the distant sites of metastasis with fatal results. The differentiation between apocrine and eccrine metastatic sweat gland carcinoma is often difficult. The criteria's are inadequate to be of any practical utility.