Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

The poison–antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison–antidote plasmid stability system. The three genes were named pasA , pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison–antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison–antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

Leilani Muir versus the Philosopher King: Eugenics on trial in Alberta

The Province of Alberta in Canada was the only jurisdiction in the British Empire where a eugenic sterilization law was passed (in 1928) and vigorously implemented. The pace of sterilization orders accelerated during the Nazi era and remained high after World War II, terminating only in 1972 when the Sexual Sterilization Act was repealed. The Alberta Eugenics Board operated away from public and legislative scrutiny, and many things done in the name of eugenics were clearly illegal. Eugenics was put on trial in Alberta in 1995 and a judge of the Court of Queen's Bench ruled in 1996 that the government had wrongly sterilized Leilani Muir. After hearing evidence about the history of the eugenics movement, the origins of Alberta's Sexual Sterilization Act, the operation of the Eugenics Board, and details of Muir's life, Madam Justice Joanne B. Veit found that ‘the damage inflicted by the operation was catastrophic’, the ‘wrongful stigmatization of Ms. Muir as a moron ⋯ has humiliated Ms. Muir every day of her life’, and ‘the circumstances of Ms. Muir's sterilization were so high-handed and so contemptuous of the statutory authority to effect sterilization, and were undertaken in an atmosphere that so little respected Ms. Muir's human dignity that the community's, and the court's, sense of decency is offended’. Veit awarded Muir damages of $740,780 CAD and legal costs of $230,000 CAD. The order for Muir's sterilization was signed by John M. MacEachran, founder of the Department of Philosophy and Psychology at the University of Alberta and chairman of the Eugenics Board from 1929 to 1965. An exponent of Platonic idealism, MacEachran believed sterilization of children with a low IQ test score was a means of ‘raising and safeguarding the purity of the race’. However, the Alberta Sterilization Act was passed and implemented with cavalier disregard for the principles of genetics as well as the rights of children. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion.

The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.     

Assembly of local communities: consequences of an optimal body size for the organization of competitively structured communities

Much empirical evidence suggests that there is an optimal body size for mammals and that this optimum is in the vicinity of l00g. This presumably reflects an underlying fitness function that is greatest at this mass. Here, I combine such a fitness function with an equilibrium model of competitive character displacement to assess the potential influence of a globally optimal body size in structuring local ecological communities. The model accurately predicts the range of body sizes and the average difference in size for species in communities of varying species richness. The model also predicts a uniform spacing of body sizes, rather than the gaps and clumps in the sizes of coexisting species observed in real communities. Alternative explanations for this phenomenon are discussed. The allometric relationships that result in a body size optimum subsume a large number of characteristics associated with the physiological, behavioral, demographic, and evolutionary dynamics of the species. Further integration of the underlying dynamics (e.g. individual energetics) of these relationships into all hierarchical levels of ecology will have to incorporate multiple interactive sites, spatial heterogeneity, and phylogenetic structure, but it has the potential to provide important discoveries into the means by which natural selection operates.     

Long-Term Drug Treatment of Obesity in a Private Practice Setting

ATKINSON, RICHARD L, ROY C BLANK, DONALD SCHUMACHER, NIKHIL V DHURANDHAR, DOUGLAS L RITCH. Long-term drug treatment of obesity in a private practice setting. This study evaluated the long-term efficacy and safety of the combination of phentermine and fenfluramine for the treatment of obesity in a private practice setting. A total of 1388 consecutive, qualified patients presenting to a private general internal medicine practice in Charlotte, NC, were enrolled with eligibility criteria including: age 18 years to 60 years, 20% over “desirable” bodyweight or body mass index <27, no serious medical or psychiatric disease, and no contraindications to drug therapy. Patients were instructed in diet, exercise, and behavior modification techniques and received phentermine (15 mg/day to 30 mg/day) and fenfluramine (20 mg/day to 60 mg/day) continuously for over 3 years. Average duration of treatment was 15. 9 months, and average weight loss at the last visit was 11. 6 kg, or 11. 7% of initial bodyweight. For patients completing 1 year of drug treatment, mean weight loss was 16. 5 kg, or 16% of initial weight. Weight loss persisted for 2 years, but partial regain was seen at 3 years. The dropout rates were 18% at 6 months, 39% at 1 year, 68% at 2 years, and 78% at 3 years. At 1 year, blood pressure of hypertensive patients fell from 151/95 mm Hg to 127/78 mm Hg, and serum cholesterol and triglycerides of hyperlipidemic patients fell by 0. 750 mmol/L (29 mg/dL) and 0. 937 mmol/L (83 mg/dL), respectively. Adverse events were modest. We conclude that, in a private practice setting, long-term treatment of obesity with the combination of phentermine, fenfluramine, and a weight maintenance program is generally safe and effective. More research is needed to determine efficacy and safety for longer than 3 years.