Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity

The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.     

Mitochondria as targets for chemotherapy

Mitochondrial malfunctioning is implicated in the pathogenesis of a variety of disorders, including cancer and multiple neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. Disturbance of mitochondrial vital functions, e.g., production of ATP, calcium buffering capacity, and generation of reactive oxygen species, can be potentially involved in disease pathogenesis. Neurological disorders caused by mitochondrial deterioration are often associated with cell loss within specific brain regions. In contrast, mitochondrial alterations in tumor cells and the “Warburg effect” might lead to cell survival and resistance of tumor cells to chemotherapy. This review is devoted to the role of mitochondria in neurodegeneration and tumor formation, and describes how targeting of mitochondria can be beneficial in the therapy of these diseases, which affect a large human population.     

Impact of exercise training on insulin sensitivity, physical fitness, and muscle oxidative capacity in first-degree relatives of type 2 diabetic patients

First-degree relatives of type 2 diabetic patients (offspring) are often characterized by insulin resistance and reduced physical fitness (VO2 max). We determined the response of healthy first-degree relatives to a standardized 10-wk exercise program compared with an age-, sex-, and body mass index-matched control group. Improvements in VO2 max (14.1 +/- 11.3 and 16.1 +/- 14.2%; both P < 0.001) and insulin sensitivity (0.6 +/- 1.4 and 1.0 +/- 2.1 mg x kg(-1) x min(-1); both P < 0.05) were comparable in offspring and control subjects. However, VO2 max and insulin sensitivity in offspring were not related at baseline as in the controls (r = 0.009, P = 0.96 vs. r = 0.67, P = 0.002). Likewise, in offspring, exercise-induced changes in VO2 max did not correlate with changes in insulin sensitivity as opposed to controls (r = 0.06, P = 0.76 vs. r = 0.57, P = 0.01). Skeletal muscle oxidative capacity tended to be lower in offspring at baseline but improved equally in both offspring and controls in response to exercise training (delta citrate synthase enzyme activity 26 vs. 20%, and delta cyclooxygenase enzyme activity 25 vs. 23%. Skeletal muscle fiber morphology and capillary density were comparable between groups at baseline and did not change significantly with exercise training. In conclusion, this study shows that first-degree relatives of type 2 diabetic patients respond normally to endurance exercise in terms of changes in VO2 max and insulin sensitivity. However, the lack of a correlation between the VO2 max and insulin sensitivity in the first-degree relatives of type 2 diabetic patients indicates that skeletal muscle adaptations are dissociated from the improvement in VO2 max. This could indicate that, in first-degree relatives, improvement of insulin sensitivity is dissociated from muscle mitochondrial functions.     

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (approximately 10%) was recovered by reductive treatment of cell protein with NaBH4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (greater than 200 microM) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.     

The effect of gossypol on the frequency of DNA-strand breaks in human leukocytes in vitro

Gossypol, a human antifertility agent isolated from the cotton plant, was found to induce a dose-dependent increase in the frequency of DNA-strand breaks in human leukocytes exposed to 2-40 micrograms/ml of the drug for 1 h in serum-free medium in vitro. DNA-strand breaks were studied by alkaline elution or alkaline unwinding of DNA followed by hydroxylapatite-chromatography. No decrease of gossypol-induced DNA-strand breaks was observed after post-treatment incubation times up to 24 h, whereas X-ray-induced DNA breaks disappeared within 2 h under the same incubation conditions. Cells exposed to gossypol in the presence of 10% fetal calf serum showed no or little increase of DNA breaks, suggesting that serum proteins inhibit the DNA-damaging activity of the drug. Both optical isomers of gossypol induced DNA-strand breaks. However, the effect of (-)-gossypol was only about half of that of (+)-gossypol and the racemic form. The induction and persistence of DNA-strand breaks by gossypol, as well as the reduction of this effect in the presence of serum should be considered in the evaluation of the potential in vivo genotoxicity of the drug.     

Distribution patterns of morph frequencies in the snail Cepaea hortensis in Iceland

In Iceland Cepaea hortensis Müll. reaches its northern limit of distribution occurring in restricted areas along the south coast. Samples of snails collected along steep cliffs in two such restricted areas were effectively monomorphic for shell colour (yellow), but were polymorphic for banding pattern and revealed strong area effects. In both localities samples from sites facing east contained varying frequencies of unbanded snails whereas in ecologically similar sites facing south unbanded were absent. In an experimental population released at a south-facing site unbanded snails failed to become established whereas banded individuals survived and bred. It is suggested that the area effects are maintained through non-visual (most likely climatic) selection though other possibilities cannot be ruled out.     

ON THE RELATIONSHIP OF LIVER GLUCOSE-6-PHOSPHATASE TO THE PROLIFERATION OF ENDOPLASMIC RETICULUM IN PHENOBARBITAL INDUCTION

The differentiated effects of phenobarbital treatment on liver microsomal enzymes have been further studied. The relationship between the resulting decrease in the specific glucose-6-phosphatase activity and the enhancement of formation of endoplasmic reticulum membranes with high drug-hydroxylating activity has been investigated with biochemical and histochemical methods. Biochemically and histochemically demonstrable glucose-6-phosphatase activity was found to be present in all endoplasmic reticulum membranes, including the phenobarbital-induced smooth-surfaced proliferates, even though there was an over-all decrease in activity. Actinomycin D did not inhibit the decrease in glucose-6-phosphatase activity. The findings are discussed with reference to the enzyme-membrane relationship in phenobarbital induction.     

Hydroxyurea,a natural metabolite and an intermediate in d-cycloserine biosynthesis in streptomyces garyphalus

It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [¹⁴C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-¹⁴C]-arginine and l-[ureido-¹⁴C]-citrulline. The ¹⁵N of H₂NCO¹⁵NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the ¹⁵N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [¹⁵N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine.     

Remodelling of triacylglycerols in microsomal preparations from developing castor bean (Ricinus communis L.) endosperm

Microsomal preparations from developing castor bean (Ricinus communis L.) endosperm catalyzed remodelling of in-situ-formed triacylglycerol (TAG) species. Castor bean microsomal membranes synthesized [¹⁴C]TAGs from either glycerol 3-phosphate and [¹⁴C]ricinoleoyl-CoA or [¹⁴C]glycerol 3-phosphate and ricinoleoyl-CoA. Upon repelleting and subsequent incubation of the microsomes a redistribution occurred of both the [¹⁴C]glycerol and [¹⁴C]ricinoleoyl moieties of the in-situ-synthesized [¹⁴C]TAGs. Radioactivity was transferred from TAG species with three (3HO-TAG) or two (2HO-TAG)ricinoleoyl groups into species with two or one (HO-TAG) ricinoleoyl groups. Mass analysis of the lipid and fatty acid movements in the membranes showed that a net synthesis of TAGs with no, one and two ricinoleoyl groups occurred at the expense of 3HO-TAG and polar lipids. Thus, the non-hydroxylated acyl groups from polar lipids were used in the remodelling of TAGs. In-vivo feeding of [¹⁴C]ricinoleic acid to slices of castor bean endosperm demonstrated the presence of two radioactive pools of TAGs one in the oil bodies, which was rich in [¹⁴C]3HO-TAG, and one associated with the microsomal membranes, which was dominated by radioactive 1HO-TAG and 2HO-TAG. The microsomal TAG pool was remodelled in vivo in a similar way as in the in-vitro experiments with microsomal membranes. Received: 8 November 1996 / Accepted: 5 February 1997     

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

The RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%-15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis.