Rosemary Lemons

Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia

The hereditary spastic paraplegias (HSPs; Strümpell-Lorrain syndrome, MIM number 18260) are a diverse class of disorders characterized by insidiously progressive lower-extremity spastic weakness (reviewed in refs. 1–3). Eight autosomal dominant HSP (ADHSP) loci have been identified, the most frequent of which is that linked to the SPG4 locus on chromosome 2p22 (found in ∼42%), followed by that linked to the SPG3A locus on chromosome 14q11–q21 (in ∼9%). Only SPG4 has been identified as a causative gene in ADHSP. Its protein (spastin) is predicted to participate in the assembly or function of nuclear protein complexes. Here we report the identification of mutations in a newly identified GTPase gene, SPG3A, in ADHSP affected individuals.

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

The loss of ATRX impairs DNA repair, promoting glioma growth but enhancing sensitivity to DNA-damaging therapies. Aggressive gliomas’ Achilles’ heel ATRX is a protein that is often mutated in glioma, a lethal and relatively common brain tumor. Koschmann et al. developed a mouse model of ATRX-deficient glioma and discovered that these tumors grow more aggressively than their counterparts with wild-type ATRX. The reason this happens is that the loss of ATRX impairs DNA repair, resulting in genetically unstable tumors that can accumulate oncogenic mutations more quickly. However, because of their DNA repair defect, these tumors also proved to be more sensitive to treatments that damage the DNA, such as radiation and some types of chemotherapy. Consistent with these findings, the presence of ATRX mutation correlated with better outcomes in patients, because these tumors were more susceptible to treatment. Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Modulation of the Intracellular Cystine Content of Cystinotic Fibroblasts by Extracellular Albumin

Summary: Cystinotic fibroblasts contain highly elevated amounts of intraellular non-protein cystine within lysosomes compared to normal fibroblasts. Both the rate of cystine reaccumulation by cystin-delepleted cystinotic fibroblasts and the steady-state cystine content of nondepleted cystinotic fibroblasts can be modulated by the addition of bovine scrum albumin to the culture medium. This effect is not seen in cultures of normal and cystinotic heterozygote fibroblasts. The cystinotic homozygote cells accumulate cystine under these conditions from proteolysis of the albumin. An increased rate of pinocytosis or proteolysis of albumin does not account for the observed cystine accumulation by the cystinotic fibroblasts. Comparison of the amount of cystine accumulated to the amount of albumin degraded shows that less than one percent of the cystine moieties released by proteolysis is retained within these cells.Speculation: The metabolic defect leading to cystine storage in cystinosis remains to be identified. Study of the relationship between the rate of cystine accumulation and the cystine and cysteine content of proteins degraded by cystinotic fibroblasts may help to determine if the defect is related to an abnormality of lysosomal disulfide reduction.

A simple method for determination of plasma and urinary biotin

Measurement of biotin in plasma and urine has been stimulated by recent descriptions of inborn errors of biotin metabolism and by newly recognized causes of biotin deficiency. Biotin determination in physiologic fluids to document these conditions has been hindered by lack of a widely useable assay. This paper presents a method which employs tritium-labelled biotin, avidin, and nitrocellulose filters to measure urinary and plasma biotin in a rapid and simple manner.

Protein Microinjection by Protease Permeabilization of Fibroblasts

Exposure of cultured diploid fibroblasts to protease solutions induces a hyperpermeable state which permits entry of exogenous macromolecules directly into the cytosol. We have exploited this finding to devise a microinjection method whose chief advantages are simplicity and good retention of cell viability. Proteins successfully injected by this technique range from insulin to thyroglobulin. The amounts injected range from 4 X 10(5) to 5 X 10(6) molecules/cell.

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

Cystine depleted cystinotic fibroblasts incubated in cystine-free medium accumulate lysosomal-free cystine from the degradation of cystine-containing intracellular and extracellular proteins. In this report we have used this characteristic of these cells to study lysosomal proteolysis. We find that inhibitors of protein synthesis (cycloheximide, emetine, and puromycin) inhibit cystine accumulation from endogenous proteins and therefore act to inhibit lysosomal proteolysis of these proteins. However, cycloheximide does not inhibit cystine accumulation derived from the degradation of the extracellular disulfide-rich proteins, albumin and RNase, but lysosomal cystine accumulation derived from insulin is inhibited by cycloheximide. We conclude that a rapidly turning over protein may be required for the lysosomal degradation of intracellular and some extracellular proteins.

Elevated temperature produces cystine depletion in cystinotic fibroblasts

Increasing the incubation temperature of cystinotic fibroblasts to 40 or 43 degrees C produces a 70-80% decrease in lysosomal cystine content within 24-48 h. This effect is probably mediated by an altered substrate affinity for another lysosomal transport protein.

Utilization of mercaptoethylgluconamide for depleting human cystinotic fibroblasts of their accumulated lysosomal cystine

ABSTRACT: Human cystinotic fibroblasts were completely depleted of their accumulated intracellular free cystine within a 2-h time interval when exposed to culture medium containing between 1 and 5 mM mercaptoethylgluconamide. This cystine-depleting action of mercaptoethylgluconamide was observed with three different human cystinotic fibroblast cell lines and with all three cell lines, 2 mM mercaptoethylgluconamide was as effective as 1 mM cysteamine in depleting cells of their intracellular free cystine. Cell viability was excellent for cystinotic fibroblasts exposed to 2 mM mercaptoethylgluconamide for up to 6 days in duration. Mercaptoethylgluconamide (2 mM) was sufficiently stable under cell culture conditions such that a single addition of mercaptoethylgluconamide maintained cystine depletion in human cystinotic fibroblasts for at least a 4-day period. In contrast to cysteamine, 2 mM mercaptoethylgluconamide was not capable of depleting the cystine content of isolated cystinotic lysosomes, implying that cellular integrity is necessary to achieve cystine depletion by mercaptoethylgluconamide. The efficient cystine-depleting action of mercaptoethylgluconamide coupled with its lack of offensive odor encourage further investigation of this agent to possibly complement or supplant the use of cysteamine in the treatment of nephropathic cystinosis.

Next generation sequencing panel based on single molecule molecular inversion probes for detecting genetic variants in children with hypopituitarism

Congenital Hypopituitarism is caused by genetic and environmental factors. Over 30 genes have been implicated in isolated and/or combined pituitary hormone deficiency. The etiology remains unknown for up to 80% of the patients, but most cases have been analyzed by limited candidate gene screening. Mutations in the PROP1 gene are the most common known cause, and the frequency of mutations in this gene varies greatly by ethnicity. We designed a custom array to assess the frequency of mutations in known hypopituitarism genes and new candidates, using single molecule molecular inversion probes sequencing (smMIPS).

FURTHER CHARACTERIZATION OF LYSOSOMAL SYSTEM y

Using a trans-stimulation property associated with lysine exodus, we have demonstrated previously a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. By studying instead the uptake of arginine into highly purified flbroblast lysosomes, obtained by centrifuging through Percoll density gradients, we now examine additional characteristics of this system. For arg uptake it displays a broad pH optimum from pH 7.0-8.0. The rate of arg uptake is 10-fold greater at pH 7.0 than 5.0, thus favoring net entry of arg into lysosomes as a result of the low intralysosomal pH. In contrast, external MgATP accelerates lysosomal efflux of cationic amino acids while inhibiting their influx. Trans-stimulation of arg uptake is seen when lysosomes have been loaded with 2-aminoethyl-L-cysteine. Arg uptake (.03 mM) is strongly inhibited by the L-isomers of external 3.3 mM arg, lys, orn, 2, 4-diaminobutyrate, 2-aminoethylcysteine and his, whereas D-arg, neutral and anionic amino acids have little effect. In addition, lysosomal arg uptake is inhibited by α-N-methyl-L-arg (72%) and e-trimethyl-L-lys (49%), neither of which are recognized by the plasma membrane System y+. These observations indicate that lysosomal System y+ is structurally different from System y+ of the plasma membrane of the human fibroblast and various other cells. Thiocholine (TC) depleted cystinotic fibroblasts of their accumulated cystine to the same level and rate as produced by cysteamine supporting the view that TC may react with cystine to form a mixed disulfide recognized by lysosomal System y+ similar to the one formed by cysteamine. Support ackn. from Grant AM32281, NIH.