A yeast system for stable expression of hepatitis B surface antigen.

We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

High biliary and systemic excretion of a glucuronide during its hepatic synthesis

Using isolated perfused rat liver and ¹⁴C-p-nitrophenol as a model substrate for glucuronidation, evidence was obtained that during synthesis of p-nitrophenyl-glucuronide biliary excretion of the metabolite is much higher than would be expected from concentrations of the metabolite in the circulation. Isotopic dilution experiments indicate that newly synthesized glucuronide is in a pool which is readily available for biliary excretion but is not easily miscible, or diluted, by the glucuronide in the circulation.     

Clinical and cytologic aspects of primary fallopian tube carcinoma. A report of ten cases

The clinical and cytologic findings in ten cases of primary fallopian tube carcinoma, a very rare malignancy, are presented. All ten patients had vaginal pool (V), cervical (C), endocervical (E) and endometrial aspiration (EA) smears examined preoperatively; peritoneal smears were also prepared from cul-de-sac fluid aspirated during surgery. In the preoperative cytodiagnosis, some of V, C, E and EA smears were positive for malignant cells in six of the ten patients (60%). Examination of the EA smear was the most effective means of discovering this disease. A watery discharge was noted in two patients, both of whom were negative in the preoperative cytodiagnosis. In such patients, it is necessary to repeat the smear examinations. The cytologic appearance of this disease is contrasted to that of endometrial carcinoma.     

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.     

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

Summary We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 m-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA contnet of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry of individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA contnet of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Precentral neuron activity associated with ipsilateral forelimb movements in monkeys.

Unit activity was recorded in motor cortex on one side, while monkeys were moving left or right fingers, wrists, or arms. On hundred and eighty five movement-related neurons were obtained from two monkeys. Of these, 122 were related to contralateral movements, 50 were to movements of both sides, and the remaining 13 to ipsilateral movements. It was found that ipsilateral-movement-related neurons tended to appear in groups of neurons that were related more to arm movements than to finger and/or wrist movements.