Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C-terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.     

Expression of the cystic fibrosis gene in human development.

The specialised epithelia lining the respiratory tract, pancreatic ducts, male genital ducts and sweat gland ducts are defective in the severe inherited disease, cystic fibrosis (CF). We have looked at the expression of the CF gene in human fetal tissues to throw light on the development of function in specialised ductal epithelia and to determine the age of onset of the CF disease process. The CF gene is already seen to be transcribed in mid-trimester fetal lung, pancreas and male genital ducts. Hence, by this developmental stage, and before they are fully differentiated, these epithelia have the capability to perform important transport functions. Epithelial cell cultures derived from fetal pancreas and male genital ducts maintain expression of the CF gene in vitro and so form good models for analysing CF gene function and differentiation of these specialised epithelia.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

An endogenous morphine-like factor in mammalian brain.

An endogenous morphine-like substance (MLF) found in rat and calf brains has a regional distribution correlating with that of opiate receptors, with the highest levels in the caudate and negligible amounts in the cerebellum. In binding assays MLF behaves like an opiate agonist. Sodium ion and enzyme and reagent treatment of membranes decrease its potency and manganese ion enhances it. MLF is localized in synaptosomal fractions, stored in an osmotically labile compartment, and can be degraded by carboxypeptidase A and leucine amino peptidase, implying a peptide structure. Its molecular weight is about 1000 as determined by gel chromatography.     

Synthetic C-nucleosides: 3-(alpha- and beta-D-arabinofuranosyl)pyrazolo(4,3-d)pyrimidine-5,7-diones

2,3,5-Tri-O-benzyl-D-arabinofuranosyl bromide (4) was converted into 2,5-anhydro-3,4,6-tri-O-benzyl-D-glucononitrile (5), mixed with 20% of the D-manno epimer 6. The mixture was reduced to the amine 7, which via the N-nitrosoacetamide 10 afforded the 1-deoxy-l-diazo sugar 11. Dipolar addition to dimethyl acetylene-dicarboxylate afforded the C-nucleoside derivative, dimethyl 3-(2,3,5-tri-O-benzyl-α-β-D-arabinofuranosyl)pyrazole-4,5-dicarboxylate (20). Selective ammonolysis afforded the 4-ester-5-carboxamide 21, which was separated chromatographically into the α-(minor) and β-(major) anomers. Hydrazinolysis and Curtius reaction of the pair of 4-acid hydrazides (α-22 and β-22) afforded the anomeric 3-glycosyl-1H-pyrazolo-[4,3-d]pyrimidine-5,7-diones (α-24 and β-24). Hydrogenolytic debenzylation yielded the β-D)-arabino epimer (1) of oxoformycin B, and the α-D-arabino form 2. These anomeric C-nucleosides were distinguished by circular dichroism spectra that showed the same relationship as α- and β-D-arabino anomers of normal purine nucleosides.