Cell cycle control and CDC28/Cdc2 homologue and related gene cloning of Cryptococcus neoformans]

In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.     

Study of the effects of dopamine hydrochloride on alpha 1-adrenoceptors in rat kidney

We studied the number and affinity of catecholamine receptors in SD rat kidney by radioreceptor technique. The following conclusions were obtained: 1) By using 3H-prazosin, the numbers of alpha 1-receptor (Bmax) in rat renal cortex were greater than those in rat kidney medulla. As for affinity (Kd), the significance was not recognized between the two. Bmax of the rat renal cortex to 3H-prazosin binding was 96.1 fmol/mg protein, and Kd was 0.17 nM, and for the rat renal medullar these values were 44.5 fmol/mg protein and 0.13 nM, respectively. 2) By measurement of D1-receptor using 3H-SKF38393 in the rat renal cortex in the Scatchard plot analysis, positive cooperativity was observed under the low concentration of hot ligand which was less than 1 nM. But at the concentration of hot ligand over 1 nM, the plots showed a straight line. Bmax of the rat renal cortex to 3H-SKF38393 was 2.5 pmol/mg protein and Kd was 5.3 nM. 3) Based on displacement by dopamine for 3H-prazosin binding to rat renal cortex, it was surmised that high concentration of dopamine had an affinity to alpha 1-adrenoceptors. 4) There was no change in the Kd and Bmax of alpha 1-receptor in the rat renal cortex after incubation of samples with low concentration of dopamine. However, in the case of high concentration of dopamine, a remarkable decrease of the affinity (Kd) of alpha 1-adrenoceptor was observed.     

l-DOPA induces concentration-dependent facilitation and inhibition of presynaptic acetylcholine release in the guinea-pig submucous plexus

Neurotransmitter- or neuromodulator-like actions ofl-DOPA were investigated with intracellular recordings from submucous plexus neurons of the guinea-pig caecum.l-DOPA at 30 nM augmented the amplitude of fast EPSPs, but did not affect depolarizations elicited by puff application of acetylcholine (ACh). The augmenting effect ofl-DOPA on the fast EPSPs was counteracted byl-DOPA methyl ester. The fast EPSPs were depressed by 10 μMl-DOPA, but transiently augmented after rinsing the drug.l-DOPA methyl ester did not affect the inhibitory action ofl-DOPA on the fast EPSPs, but antagonized the potentiation following the inhibition. The depolarization elicited by exogenously applied. ACh was inhibited by 10 μMl-DOPA. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of the neuronal soma were measured with fura-2 microfluorophotometry. The transient increase in the [Ca²⁺]_i evoked by the somatic action potential (Δ[Ca²⁺]_AP) was facilitated by 30 nMl-DOPA, but decreased by the drug at 10 μM. It is concluded thatl-DOPA at low concentrations enhances the Δ[Ca²⁺]_AP, increasing the neurotransmitter release, but at high dose diminishes the Δ[Ca²⁺]_AP, inhibiting the neurotransmission.     

Biodistributions of 201Tl in tumor bearing animals and inflammatory lesion induced animals

The accumulation of 201Tl in tumor and inflammatory tissues were small. However, this nuclide showed a high concentration in viable tumor tissue, less in connective tissue (containing inflammatory tissue), and was not seen in necrotic tumor tissue regardless of the time after administration of 201Tl(I)-chloride. In inflammatory lesions, 201Tl accumulated in subcutaneous tissue infiltrated with neutrophils and macrophages, and quite large amounts of this nuclide were accumulated in subcutaneous tissue and sites where neutrophils were crowded. Most 201Tl existed in a free form in the fluid of tumor and inflammatory tissues regardless of the time after administration. A small amount of this nuclide was localized in the nuclear, mitochondrial and microsomal fractions in these tissues, and the nuclide was bound to protein in these fractions. The distribution of 201Tl(III)-chloride in tumor bearing animals was essentially the same as that of 201Tl(I)-chloride.     

Evidence for a defect in urinary concentrating ability in primary aldosteronism and its reversal by adrenal surgery

Of 15 patients with primary aldosteronism, 7 had idiopathic adrenal hyperplasia (IHA) and 8 had aldosterone-producing adenoma (APA). In order to determine any renal problems involved in the treatment, the renal clearance of these patients was analyzed and the results compared with those obtained from 12 patients with essential hypertension. With water diuresis or under antidiuresis status, levels of urine volume, Cosm and CH2O in patients with APA were greater (p less than 0.05-p less than 0.001) than those of patients with essential hypertension, while the fractional tubular sodium delivery of the former patients was lower than that of the latter patients (p less than 0.001 or less than 0.05). A similar tendency was observed in clearance studies in patients with IHA, although to a lesser extent. Adrenal surgery for patients with APA normalized these values, but administration of trilostane (3 beta-hydrosteroid dehydrogenase inhibitor) to patients with IHA failed to improve these values. These results indicate that impaired urinary concentrating ability as well as reduced urinary diluting capability is a common feature of primary aldosteronism. Such impaired renal function was improved only in patients with APA after adrenal surgery.