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Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.

A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

The androgen resistance syndromes are generally felt to be due to quantitative or qualitative abnormalities of the androgen receptor. Some patients with testicular feminization have no demonstrable fibroblast cytosol androgen binding, whereas others have androgen binding in cultured fibrobalsts that is thermolabile or fails to be stabilized by sodium molybdate. I describe here familial incomplete testicular feminization associated with reduced nuclear androgen retention. Fibroblasts, cultured from pubic skin biopsies of two phenotypic female 46XY siblings, were assayed for whole cell and nuclear uptake of [(3)H]dihydrotestosterone in dispersed, intact cells. Whole cell binding of [(3)H]dihydrotestosterone at 22 degrees C in the patients' fibroblasts was in the normal range. However, no high affinity, saturable binding of [(3)H]dihydrotestosterone was demonstrable in crude nuclear pellets prepared from the patients' fibroblasts incubated at 37 degrees C with the hormone. Incubating the patients' cells with [(3)H]methyltrienolone or examining the nuclear uptake of [(3)H]dihydrotestosterone in these cells at 22 degrees C did not alter these findings. Although cytosol from the patients' cells revealed a quantitatively diminished 8S peak for [(3)H]dihydrotestosterone after centrifugation on sodium molybdate-containing sucrose gradients, there was no peak of (3)H in the 4S region from 0.3 M KCl nuclear extracts of the patients' cells after they had been incubated with [(3)H]dihydrotestosterone at 37 degrees C. Although whole cell binding studies at 37 degrees C showed minimally diminished androgen binding in the patients' cells compared with binding at 22 degrees C, Griffin (1979. J. Clin. Invest.64: 1624-1631.) has demonstrated thermolability of the androgen receptors in fibroblasts also cultured from these patients. The observations with intact cells coupled with the diminished cytosol 8S peak of [(3)H]dihydrotestosterone on sucrose gradients indicate that these patients have cytosol androgen receptors that are qualitatively abnormal physicochemically, the physiologic consequence of which is failure of nuclear androgen localization. Thus, although the underlying defect in the pathogenesis of the androgen resistance in these patients appears to reside in the androgen receptor, the crucial biologic manifestation of the molecular lesion is impaired nuclear androgen retention. These experiments, therefore, suggest that assessment of nuclear [(3)H]dihydrotestosterone uptake is an effective indicator of the functional integrity of the androgen receptor system in patients with various forms of androgen insensitivity and provides additional insights to those obtained by thermolability or cytosol sucrose gradient studies.

Cryostat sections of unfixed human breast carcinomas were assayed for oestrogen receptor (ER) content using an isoelectric focusing method to separate the receptor-bound oestradiol. Adjacent sections from the same tissue block were stained so that the tumour content could be estimated by point counting and the ER concentration adjusted to compensate for variations in cellularity. Elastosis was also assessed. The results confirm a positive correlation between ER values and both cellularity and elastosis. The measurement of ER in cryostat sections is relatively simple and rapid, is applicable to small tissue samples, and permits histological identification of the nature and composition of the assayed sample. The method is directly applicable to oestrogen receptor analyses of breast carcinomas in clinical laboratories with facilities for cryostat microtomy.

Chromium(III) has been reported to be an essential trace element involved in the control of glucose metabolism by insulin. We have studied the distribution and kinetics of intravenous [51Cr]chromium(III) in six human subjects using a whole-body scintillation scanner, a whole-body counter, and plasma counting. Principal concentrations were found in the liver, spleen, soft tissue, and bone. The data were fit to a model consisting of a plasma pool in equilibrium with fast (T1/2 = 0.5-12 h), medium (1-14 days), and slow (3-12 mo) compartments, and transfer rates were calculated for exchanges between compartments. Each of the imaged organs appeared to contain varying proportions of each compartment. Two patients with hemochromatosis, in which iron overload is postulated to exclude chromium transport, were found to have altered rate constants. The results should be useful in assessing the metabolism and nutritional requirement of chromium(III) in humans and its importance in disease processes such as diabetes.

Parenteral administration of iron as the complex with nitrilotriacetate resulted in moderate iron deposition in the renal cortex along with heavy deposition of iron in liver, heart, and exocrine pancreas. Iron treated rats exhibited slight but significant changes in serum chemistries consistent with mild renal insufficiency: increased chloride and creatinine and decreased sodium, calcium and CO2 content as a group with isolated increases in urea nitrogen (2/9) and phosphorus (3/9). Serum albumin was decreased and the normal correlation of serum calcium with serum albumin was abolished by iron overload. Although food intake/g body weight was similar in the two groups, both water intake and urine output relative to solute load were increased in iron-overloaded rats. We conclude that the renal insufficiency occasionally reported in human hemochromatosis may reflect a direct nephrotoxic action of iron.