Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.     

Seeds to crystals

Seeding has been critical for obtaining diffraction-quality crystals for many structures. In this article, applications and recommendations for seeding are presented based on examples from our laboratory and other groups. The implementation of seeding in high-throughput crystallization, robotics, and other emerging technologies is also discussed.     

Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.     

Biochemical and Immunocytochemical Evidence for a Deficiency of Normal Interfascicular Oligodendroglia in the CNS of the Dysmyelinating Mutant (md) Rat

Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant.     

Genetics of methane and methanol oxidation in Gram-negative methylotrophic bacteria

Within the past few years, considerable progress has been made in the understanding of the molecular genetics of methane and methanol oxidation. In order to summarize this progress and to illustrate the important genetic methods employed, this review will focus on several well-studied organisms. These organisms include the gramnegative faculative methylotrophsMethylobacterium extorquens, Methylobacterium organophilum andParacoccus denitrificans. In addition, the obligate methanotrophsMethylococcus capsulatus andMethylosinus trichosporium are discussed. We have chosen not to discuss the genetics of methanol oxidation in the yeasts or in gram-positive bacteria. Likewise, the genetics of related topics (for example, methylamine oxidation and carbon assimilation pathways) are not reviewed here. Broad host range conjugatable plasmids have enabled researchers to complement mutations and clone genes from gram-negative methylotrophic bacteria. More recently, promoter probe derivative plasmids have been used to elucidate aspects of gene regulation. Also, alternative gene-cloning techniques are proving useful in circumventing problems in the genetic studies of the obligate methanotrophs, the group of bacteria that is the most refractory to traditional methods.     

Small differences in Drosophila tropomyosin expression have significant effects on muscle function.

The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.