Short-term metabolic fate of [13N]ammonia in rat liver in vivo

The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.     

Mortality among male anaesthetists in the United Kingdom, 1957-83.

A cohort of 3769 male anaesthetists resident in the United Kingdom between 1957 and 1983 was followed up for a total of 51,431 person years of observation. All subjects were fellows of the Faculty of Anaesthetists and held full registration with the General Medical Council. With all men in social class I being taken as the standard, the standardised mortality ratio among anaesthetists for all causes of death was 68 (95% confidence interval 59 to 77) and the standardised mortality ratio for all cancers was 50 (95% confidence interval 36 to 67). There was no significant excess mortality from lymphomas or leukaemias, but 16 of the 221 deaths in anaesthetists were due to suicide, giving a standardised mortality ratio of 202 (95% confidence interval 115 to 328). When anaesthetists were compared with all doctors the standardised mortality ratio for suicide was only 114, a nonsignificant excess. These findings confirm that the risk of suicide among anaesthetists is twice as high as among other men in social class I but suggest that the risk does not differ significantly from that among doctors as a whole. There was no evidence of a significant excess risk of cancer, and, in particular, the small excess of cancer of the pancreas reported previously could not be confirmed.     

Jejunal free autograft: analysis of complications and their resolution

Review of 101 patients who underwent 111 free jejunal autografts has demonstrated an absolute procedural failure rate of 13.5 percent. Salvage reconstruction with a second jejunum was successful in six of nine patients and one third-time jejunum was successful, giving an overall salvage rate of 70 percent. There were 33 patients experiencing pharyngocutaneous fistulas, 20 of whom had been previously irradiated. Of these patients, 15 experienced spontaneous closure and 9 others had successful surgical correction. The mortality rate was 5 percent. Eighty-three percent of patients were restored to adequate per oral alimentation. The jejunum, despite its relatively high complication rate, is an excellent method for pharyngoesophageal reconstruction, expeditiously providing return to function for patients with late-stage disease.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system.

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.     

1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ambient temperature cycles entrain the free-running circadian rhythms of the stripe-faced dunnart,Sminthopsis macroura

Summary We examined the effect of cycles of 12 h warm (35 ± 2 °C) and 12 h (21 ± 2 °C) ambient temperature (Ta) upon the circadian activity rhythms of stripe-faced dunnarts, Sminthopsis macroura, free-running in conditions of constant dark (DD) or constant light (LL). It was hypothesized that dunnarts would entrain to the temperature cycles (TaHLs) or show perturbations of period, and that LL would act synergistically with the TaHLs in these effects. Under DD, 2 of 6 animals showed clear entrainment to the TaHLs. Other animals exhibited changes of period () and heavy negative masking of activity during the warm fraction of the TaHLs. Under LL, 3 of 12 animals entrained to the TaHLs. It was concluded that Ta is a significant though weak Zeitgeber for S. macroura compared to light. It is possible that TaHLs entrain homeotherm activity rhythms by altering the rhythm of body temperature, which is usually tightly coupled to activity.Abbreviations TaHL a cycle of Higher and Lower ambient temperature - TaC Constant Ta - Tb body temperature