Identification and characterization of the non-PTS fru locus of Bacillus megaterium ATCC 14581.

A genetic locus that is adjacent to the gene encoding the small acid-soluble protein SASP C-4 of Bacillus megaterium has been identified. This locus, designated fru, contains a beta-fructosidase gene (fruA), a gene encoding a hydrophobic protein that is closely related to non-PTS sugar permeases of the proton symport type (fruP), and a gene coding for a transcriptional regulator of the LacI/GalR family (fruR). The FruA protein can hydrolyze sucrose and raffinose, but not maltose, isomaltose, trehalose, melibiose or lactose. The transcription initiation site of fruP has been mapped and the fruP promoter identified. Gel mobility shift assays showed that the FruR protein can bind specifically to a DNA fragment containing the fruP promoter region. DNase I footprinting analysis has defined the FruR binding site. Disruption of fruR led to high-level constitutive expression of fruPA, but had no effect on expression from the fruR promoter itself, indicating that FruR acts as a repressor of fruPA expression, but does not autoregulate its own synthesis. Interestingly, expression of fruPA in B. megaterium was not induced by sucrose, raffinose, fructose or inulin, whereas the constitutive expression of fruPA in a fruR mutant was repressed by both glucose and sucrose. Possible physiological implications of these findings are discussed.

Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581.

A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).

Study of the rat adrenal renin-angiotensin system at a cellular level.

To address the question as to how zona glomerulosa (ZG) cell angiotensin II (Ang II) secretion is regulated, we developed an immuno-cell blot assay to measure its secretion from single cells. We compared these results with those obtained from population studies using a superfusion system. Modulation of Ang II secretion was investigated acutely (by administrating potassium [K+] or captopril) and chronically (by feeding the animals low or high sodium diets). The area of secretory cells, halo areas, and halo intensities varied widely but were highly significantly correlated (P < 0.001) with each other. A disproportionate amount of Ang II was secreted by a small number of large cells. When K+ concentration was increased from 3.6 to 0 mM, superfused ZG cells increased their Ang II secretion 2.32 +/- 0.59-fold. Administration of captopril reduced the K(+)-stimulated Ang II secretion 1.24 +/- 0.07 fold. These findings were reflected in the cell blot assay as a change in the frequency distribution of halo area by K+ and captopril in the same direction as in the population study. In both conditions, the percentage of secretory cells did not change significantly from control. Superfused ZG cells from rats on a low sodium diet secreted 1.85 +/- 0.58-fold more Ang II than cells from sodium-loaded rats (p < 0.05, n = 6). The cell blot assay confirmed these findings with sodium restriction significantly increasing (P < 0.001) both the halo area and its frequency distribution to a larger portion of high secreting cells. However, in contrast to acute treatment with K+ or captopril, the number of secretory cells also doubled. Thus, the individual ZG cell uses two mechanisms to modify Ang II production. In response to acute stimulation and suppression, the amount of Ang II secreted per cell is modified without changing the number of secretary cells. With chronic stimulation, both the amount of Ang II secreted per cell and the number of secretary cells increase.

The effect of losartan on potassium-stimulated aldosterone secretion in vitro.

Potassium (K+) and angiotensin-II (Ang-II) are two distinct secretagogues for aldosterone release. However, a local adrenal renin-angiotensin system is present, and several studies suggest a complex interaction between K+ and locally produced Ang-II. First, superfusing zona glomerulosa (ZG) cells with K+ stimulates the secretion of both Ang-II and aldosterone. Second, K(+)-stimulated aldosterone secretion can be reduced in the presence of angiotensin-converting enzyme inhibitors. Because angiotensin-converting enzyme inhibitors are not specific inhibitors of the adrenal renin-angiotensin system, we further tested the hypothesis that locally produced Ang-II participates in K(+)-stimulated aldosterone release from rat ZG cells by using a specific Ang-II antagonist. Although type 1 (AT1) and type 2 (AT2) Ang-II receptors are present in ZG cells, only AT1 antagonist has been shown to mediate Ang-II-induced aldosterone secretion. Losartan, a specific AT1 antagonist, was used in this study. In the presence of losartan (10 microM for 9 mM K+ and 100 microM for 5 mM K+), the average aldosterone secretion during 2 h of superfusion with 9 mM K+ and 5 mM K+ was 70.1 +/- 5.4% (n = 5) and 58.5 +/- 2.2% (n = 3), respectively, of that in its absence. Losartan did not alter the amount of Ang-II secreted. The inhibitory effect of losartan lasted longer than 60 min after it was terminated. In summary, our results support the hypothesis that locally produced Ang-II contributes to the aldosterone secretory response to K+ stimulation at both physiological and supraphysiological levels.

Therapeutic embolization of post-cholecystectomy hepatic artery aneurysm.

A "sandwich" technique employing steel coils and gelfoam embolization was applied in two cases of hepatic artery aneurysms. Immediate cessation of the bleeding was evident clinically and the post-embolization angiogram showed occlusion of the vascular lesion. Cholecystectomy is one of the most common abdominal operations and it is generally well tolerated, particularly in young and middle-aged patients. Morbidity rates of 5% to 32% and mortality rates of 0.4% to 2.5% have been reported, depending on such factors as age, underlying illness, etc. (1-4). The most frequent complications of cholecystectomy are wound infection, abscess, ductal injury or ligation, and bleeding. This paper describes two cases of hepatic pseudoaneurysm following iatrogenic damage during cholecystectomy which was treated with transarterial embolization.

[The relationship between sexual maturation and growth of the subcutaneous sarcoma-180 tumor].

The sex hormones of mice have effects on the immune response and the synthesis of epidermal growth factor(EGF). Androgen inhibits the former and enhances the later, whereas estrogen has the opposite effect. EGF is one of the tumor growth factors. In the present study, mice before or after sexual maturation, or gonadectomized adults of both sexes, were subcutaneously inoculated with sarcoma-180 tumor cells. The tumors grew rapidly, with no difference in genders before sexual maturation. However, tumor growth was rapid in adult male mice after sexual maturation, but retarded or inhibited in adult female mice. In orchidectomized male mice, tumor growth slowed down, in ovariectomized female mice, tumor growth accelerated. There was no difference in the rate of tumor growth among gonadectomized mice of either sex.

Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically dispersed cells of the zona glomerulosa.

The cells of the adrenal cortex contain angiotensin-II (AII), but whether this peptide is synthesized there (vs. internalized from the systemic circulation), whether it is secreted, and whether it is important in aldosterone production remain uncertain. To address these issues, we studied AI and AII release from superfused rat adrenal capsules and dispersed glomerulosa cells. Superfused adrenal capsules released 7-fold more AII in 270 min than the capsules originally contained (495 +/- 101 fmol AII/rat released vs. 66 +/- 8 fmol AII/rat tissue content). The amount of AI released in the same period only slightly exceeded the tissue content. In response to higher potassium concentrations in the medium (9 vs. 3.6 mM K+), adrenal capsules and dispersed glomerulosa cells both released significantly more AI and AII into the superfusate. This release of AI and AII was oscillatory. The oscillations occurred in each of 15 experiments, with a period of 45-90 min. Decapsulated adrenal glands (the zona faciculata/reticularis plus medulla) also contained and released AII, but did not respond to potassium stimulation. There was a highly significant correlation between AII and aldosterone release. This was especially apparent if aldosterone secretion was examined during oscillations of AII release (r = 0.97; P less than 0.0001). We conclude that AII is synthesized in the zona glomerulosa and can be released in response to stimuli. The close correlation between AII and aldosterone secretion suggests that locally produced AII may play an important role in aldosterone biosynthesis.

Effects of dietary calcium on blood pressure, vascular reactivity and vascular smooth muscle calcium efflux rate in Zucker rats.

Previous data from this laboratory indicate that hypertension in insulin resistant Zucker obese rats is accompanied by an impairment in vascular smooth muscle Ca2+ efflux. Since insulin resistant states are also generally salt-sensitive and dietary Ca2+ reduces blood pressure in some salt-sensitive states, we evaluated the effects of dietary Ca2+ on blood pressure and vascular reactivity and examined whether these effects are due to increased vascular smooth muscle Ca2+ efflux. We assigned 16 obese and 16 lean rats to a normal (0.5%) or high (1.5%) Ca2+ diet for 28 days, following which intraarterial blood pressure and in vitro vascular smooth muscle 45Ca efflux and vascular reactivity responses to phenylephrine and serotonin were measured. Blood pressure was elevated in the obese rats on both diets (P less than 0.2), and the high calcium diet lowered both systolic and diastolic pressure in both the lean and obese rats (P less than 0.5). Vascular reactivity was higher in the obese rats (P less than 0.2), but dietary Ca2+ exerted opposite effects on vascular reactivity to the agonists. High Ca2+ reduced sensitivity to serotonin in the obese rats by 54% (P less than .05) without affecting sensitivity in the lean rats. In contrast, the high Ca2+ diet increased sensitivity to phenylephrine by 31% in both groups (P less than .01). 45Ca efflux was lower in the obese rats compared to the lean rats (P less than .05), and the high Ca2+ diet increased this rate by 23% in the lean, but not the obese, rats (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of sodium intake on angiotensin content of the rat adrenal gland.

To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. We conclude that these sodium-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction.

Morphological study of granular convoluted tubules in the submandibular gland of the mouse during the growth of a sarcoma-180 subcutaneous tumor.

Histological and cytological changes in the submandibular glands of adult male mice arising during the growth of sarcoma-180 subcutaneous tumors were studied. The submandibular glands of the mice were examined by morphometric analysis at 1, 3, 6, 10, 20, 30 and 64 days after inoculation of the tumor cells. There was a slow increase in the relative cross-sectional area of the granular convoluted tubule (GCT) in the section of the submandibular gland of the animals as the tumors grew. The increased proportional area of the GCT was significantly different from that of the control's from day 30. However, the mean weight of the glands was not increased. The proportional area of the granular cluster in the cells of the GCT of tumor cells in inoculated animals decreased about 5% on the first day and then quickly increased by 16% on the third day in comparison with those of the controls, eventually reaching a maximum of 74% (control, 54%) by day 30. In addition, the average number of granules per GCT cell decreased in the first three days, then increased to normal levels from day 6, going above the normal level from day 20 of the tumor growth. These changes in the glands of tumor-bearing animals disappeared within 20 days after removal of the tumor. These results indicate that the growth of the sarcoma-180 subcutaneous tumor caused morphological changes in the GCT and GCT cells, suggesting an alternation in the requirements of the secretions contained in the granules, such as the epidermal growth factor, during the growth of the tumor.

Reduction of intraocular pressure in glaucomatous dogs by a new cholinergic drug.

N-Demethylated carbachol (DMC) was synthesized and analyzed for its ocular effects. DMC has been found to penetrate ocular tissue effectively. It has been shown to be free of acute toxic properties in rabbits and dogs at therapeutic doses and has been demonstrated to lower intraocular pressure of glaucomatous beagles without causing intense miosis. The effects of DMC and pilocarpine on outflow facility were compared in normal dogs. At the dose used in these experiments, both drugs affected outflow facility to a similar extent. It is concluded that DMC could be a potentially useful ocular hypotensive agent.

Effects of diethylcholine in two animal models of Parkinsonism tremors.

(2-Hydroxyethyl) methyldiethylammonium iodide (diethylcholine; DEC) was tested against trihexyphenidyl for its ability to block tremors in two animal models of Parkinsonism tremors. Both DEC (75 mg/kg) and trihexyphenidyl (10 mg/kg) antagonized physostigmine tremors in mice. Both drugs also blocked tremors in rats which received intracaudate injections of carbachol. DEC was more efficacious than trihexyphenidyl in the rat model. No dose-related inhibition of tremors was seen for trihexyphenidyl (5--20 mg/kg) but inhibition by DEC was dose-related (25--50 mg/kg). The ED50 for tremor inhibition in the rat model by DEC was 33 mg/kg. DEC was also shown to cross the blood-brain barrier in mice. The probable mechanism of action of DEC is discussed.