Abnormal transport along the lysosomal pathway in Mucolipidosis, type IV disease

Mucolipidosis, type IV (ML-IV) is an autosomal recessive storage disease that is characterized by lysosomal accumulation of sphingolipids, phospholipids, and acid mucopolysaccharides. Unlike most other storage diseases, the lysosomal hydrolases participating in the catabolism of the stored molecules appear to be normal. In the present study, we examined the hypothesis that the ML-IV phenotype might arise from abnormal transport along the lysosomal pathway. By using various markers for endocytosis, we found that plasma membrane internalization and recycling were nearly identical in ML-IV and normal fibroblasts. A fluorescent analog of lactosylceramide (LacCer) was used to study plasma membrane lipid internalization and subsequent transport. Lipid internalization at 19°C was similar in both cell types; however, 40–60 min after raising the temperature to 37°C, the fluorescent lipid accumulated in the lysosomes of ML-IV cells but was mainly concentrated at the Golgi complex of normal fibroblasts. Biochemical studies demonstrated that at these time points, hydrolysis of the lipid analog was minimal (∼7%) in both cell types. A fluorescence ratio imaging assay was developed to monitor accumulation of fluorescent LacCer in the lysosomes and showed that the apparent concentration of the lipid increased more rapidly and to a greater extent in ML-IV cells than in normal fibroblasts. By 60 min, LacCer apparently decreased in the lysosomes of normal fibroblasts but not in ML-IV cells, suggesting that lipid efflux from the lysosomes was also impaired. These results demonstrate that there is a defect in ML-IV fibroblasts that affects membrane sorting and/or late steps of endocytosis.

Genetic influence on the structural variations of the abnormal prion protein

Prion diseases are characterized by the presence of the abnormal prion protein PrPSc, which is believed to be generated by the conversion of the α-helical structure that predominates in the normal PrP isoform into a β-sheet structure resistant to proteinase K (PK). In human prion diseases, two major types of PrPSc, type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrPSc in 36 cases of prion diseases. We demonstrated two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrPSc, respectively, and numerous secondary cleavages distributed along the region spanning residues 74–102. Accordingly, we identify three regions in PrPSc: one N-terminal (residues 23–73) that is invariably PK-sensitive, one C-terminal (residues 103–231) that is invariably PK-resistant, and a third variable region (residues 74–102) where the site of the PK cleavage, likely reflecting the extent of the β-sheet structure, varies mostly as a function of the PrP genotype at codon 129.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-d-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II

The classically recognized functions of the renin–angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b −/−) and both AT1A and AT1B receptors (Agtr1a −/−Agtr1b −/−). Agtr1b −/− mice are healthy, without an abnormal phenotype. In contrast, Agtr1a −/−Agtr1b −/− mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt−/−) and angiotensin-converting enzyme-deficient (Ace −/−) mice that are unable to synthesize angiotensin II. Agtr1a −/−Agtr1b −/− mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a −/− Agtr1b −/− mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin–angiotensin system.

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110–Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

National policies for technical change: Where are the increasing returns to economic research?

Improvements over the past 30 years in statistical data, analysis, and related theory have strengthened the basis for science and technology policy by confirming the importance of technical change in national economic performance. But two important features of scientific and technological activities in the Organization for Economic Cooperation and Development countries are still not addressed adequately in mainstream economics: (i) the justification of public funding for basic research and (ii) persistent international differences in investment in research and development and related activities. In addition, one major gap is now emerging in our systems of empirical measurement—the development of software technology, especially in the service sector. There are therefore dangers of diminishing returns to the usefulness of economic research, which continues to rely completely on established theory and established statistical sources. Alternative propositions that deserve serious consideration are: (i) the economic usefulness of basic research is in the provision of (mainly tacit) skills rather than codified and applicable information; (ii) in developing and exploiting technological opportunities, institutional competencies are just as important as the incentive structures that they face; and (iii) software technology developed in traditional service sectors may now be a more important locus of technical change than software technology developed in “high-tech” manufacturing.

Are the returns to technological change in health care declining?

Whether the U.S. health care system supports too much technological change—so that new technologies of low value are adopted, or worthwhile technologies become overused—is a controversial question. This paper analyzes the marginal value of technological change for elderly heart attack patients in 1984–1990. It estimates the additional benefits and costs of treatment by hospitals that are likely to adopt new technologies first or use them most intensively. If the overall value of the additional treatments is declining, then the benefits of treatment by such intensive hospitals relative to other hospitals should decline, and the additional costs of treatment by such hospitals should rise. To account for unmeasured changes in patient mix across hospitals that might bias the results, instrumental–variables methods are used to estimate the incremental mortality benefits and costs. The results do not support the view that the returns to technological change are declining. However, the incremental value of treatment by intensive hospitals is low throughout the study period, supporting the view that new technologies are overused.

Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.

Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells.

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.

Arabidopsis plants transformed with an antisense construct of an Arabidopsis methyltransferase cDNA (METI) have reduced cytosine methylation in CG dinucleotides. Methylation levels in progeny of five independent transformants ranged from 10% to 100% of the wild type. Removal of the antisense construct by segregation in sexual crosses did not fully restore methylation patterns in the progeny, indicating that methylation patterns are subject to meiotic inheritance in Arabidopsis. Plants with decreased methylation displayed a number of phenotypic and developmental abnormalities, including reduced apical dominance, smaller plant size, altered leaf size and shape, decreased fertility, and altered flowering time. Floral organs showed homeotic transformations that were associated with ectopic expression of the floral homeotic genes AGAMOUS and APETALA3 in leaf tissue. These observations suggest that DNA methylation plays an important role in regulating many developmental pathways in plants and that the developmental abnormalities seen in the methyltransferase antisense plants may be due to dysregulation of gene expression.

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.