The water permeability of toad urinary bladder. II. The value of Pf/Pd(w) for the antidiuretic hormone-induced water permeation pathway

Using the methods described in the preceding paper (Levine et al., 1984) for measuring the magnitude of the water-permeable barriers in series with the luminal membrane, we correct measured values of Pd(w) in bladders stimulated with low doses of antidiuretic hormone (ADH) or 8-bromo cyclic AMP to obtain their true values in the luminal membrane. Simultaneously, we also determine Pf. We thus are able to calculate Pf/Pd(w) for the hormone-induced water permeation pathway in the luminal membrane. Our finding is that Pf/Pd(w) approximately equal to 17. Two channel models consistent both with this value and the impermeability of the ADH-induced water permeation pathway to small nonelectrolytes are: (a) a long (approximately equal to 50 A), small- radius (approximately equal to 2 A) pore through which 17 water molecules pass in single-file array, and (b) a shower-head-like structure in which the stem is long and of large radius (approximately equal to 20 A) and the cap has numerous short, small-radius (approximately equal to 2 A) pores. A third possibility is that whereas the selective permeability to H2O results from small-radius (approximately equal to 2 A) pores, the large value of Pf/Pd(w) arises from their location in the walls of long tubular vesicles (approximately 2 micron in length and 0.1 micron in diameter) that are functionally part of the luminal membrane after having fused with it. Aggregate-containing tubular vesicles of these dimensions have been reported to fuse with the luminal membrane in response to ADH stimulation and have been implicated in the ADH-induced hydroosmotic response.     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

Punch shear testing of extracted vital and endodontically treated teeth

Plano-parallel specimens of human dentin cut from vital and endodontically treated teeth were tested by the punch shear test. Shear strength values were found to positively correlate with approximate toughness values. Statistically significant differences were found between shear strength and toughness values for vital and endodontically treated teeth, the latter showing lower values. The clinical impression that endodontically treated teeth are weaker and more brittle than vital teeth has therefore been quantitated. Anatomically different teeth or the methods used to store and cut teeth could not be consistently correlated with punch shear and toughness values. When dentin slices were constrained during punching so that bending was prevented, the precision of the results was improved and higher values were recorded.     

Bifurcating periodic solutions for a class of age-structured predator-prey systems

A system of mixed integrodifferential and partial differential equations for an agestructured predator-prey system is studied here. The predator eats all ages of prey, but more of the very young and very old than of the intermediate ages. The existence of periodic solutions corresponding to stable coexistence is proved for a suitable range of parameters by bifurcation theory.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Experimental allergic encephalomyelitis in congenic strains of mice

Inbred mice and lines congenic to them for the major histocompatibility complex were similar in susceptibility to EAE except for moderate differences in two pairs. TheH-2 haplotypesq, s, andb occurred in inbred strains and in congenic lines of high, medium, or low susceptibility. It is concluded that the major histocompatibility complex does not control susceptibility to EAE in mice. Furthermore, the low susceptibility of DBA/1J mice was not enhanced by poly A-U.