Augmentation of angiotensinogen expression in the proximal tubule by intracellular angiotensin II via AT1a/MAPK/NF-кB signaling pathways.

Long-term angiotensin II (ANG II) infusion significantly increases ANG II levels in the kidney through two major mechanisms: AT1 receptor-mediated augmentation of angiotensinogen (AGT) expression and uptake of circulating ANG II by the proximal tubules. However, it is not known whether intracellular ANG II stimulates AGT expression in the proximal tubule. In the present study, we overexpressed an intracellular cyan fluorescent ANG II fusion protein (Ad-sglt2-ECFP/ANG II) selectively in the proximal tubule of rats and mice using the sodium and glucose cotransporter 2 (sglt2) promoter. AGT mRNA and protein expression in the renal cortex and 24-h urinary AGT excretion were determined 4 wk following overexpression of ECFP/ANG II in the proximal tubule. Systolic blood pressure was significantly increased with a small antinatriuretic effect in rats and mice with proximal tubule-selective expression of ECFP/ANG II (P < 0.01). AGT mRNA and protein expression in the cortex were increased by >1.5-fold and 61 ± 16% (P < 0.05), whereas urinary AGT excretion was increased from 48.7 ± 5.7 (n = 13) to 102 ± 13.5 (n = 13) ng/24 h (P < 0.05). However, plasma AGT, renin activity, and ANG II levels remained unaltered by ECFP/ANG II. The increased AGT mRNA and protein expressions in the cortex by ECFP/ANG II were blocked in AT1a-knockout (KO) mice. Studies in cultured mouse proximal tubule cells demonstrated involvement of AT1a receptor/MAP kinases/NF-кB signaling pathways. These results indicate that intracellular ANG II stimulates AGT expression in the proximal tubules, leading to increased AGT formation and secretion into the tubular fluid, which contributes to ANG II-dependent hypertension.

Aberrant activation of the intrarenal renin-angiotensin system in the developing kidneys of type 2 diabetic rats.

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.

Activated intrarenal reactive oxygen species and renin angiotensin system in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is recognized worldwide as the most common primary glomerulopathy. Although the mechanisms underlying the development of IgA nephropathy are gradually being clarified, their details remain unclear, and a radical cure for this condition has not yet been established. It has been clinically demonstrated that the immunoreactivities of intrarenal heme oxygenase-1 (HO-1) and 4-hydroxy-2-nonenal (4-HNE) markers of reactive oxygen species (ROS) and those of intrarenal angiotensinogen (AGT) and angiotensin II (Ang II) markers of renin angiotensin system (RAS) in IgA nephropathy patients were significantly increased as compared to those of control subjects. In an animal study, high IgA of ddY (HIGA) mice were used as an IgA nephropathy model and compared with BALB/c mice, which served as the control. The levels of markers for ROS (urinary 8-isoprostane and intrarenal 4-HNE), RAS (intrarenal AGT and Ang II), and renal damage in the HIGA mice were significantly increased as compared to those in the BALB/c mice. Moreover, an interventional study using HIGA mice demonstrated that the expressions of 2 lines of intrarenal ROS markers (4-HNE and HO-1), 2 lines of intrarenal RAS markers (AGT and Ang II) and renal damage decreased significantly in HIGA mice receiving treatment with the Ang II receptor blocker olmesartan but not in HIGA mice receiving treatment with RAS-independent antihypertensive drugs (hydralazine, reserpine, and hydrochlorothiazide) when compared with HIGA mice that were not treated. These data suggest that intrarenal ROS and RAS activation plays a pivotal role in the development of IgA nephropathy.

Effects of Direct Renin Blockade on Renal & Systemic Hemodynamics and on RAAS Activity, in Weight Excess and Hypertension: A Randomized Clinical Trial.

AIM: The combination of weight excess and hypertension significantly contributes to cardiovascular risk and progressive kidney damage. An unfavorable renal hemodynamic profile is thought to contribute to this increased risk and may be ameliorated by direct renin inhibition (DRI). The aim of this trial was to assess the effect of DRI on renal and systemic hemodynamics and on RAAS activity, in men with weight excess and hypertension. METHODS: A randomized, double-blind, cross-over clinical trial to determine the effect of DRI (aliskiren 300 mg/day), with angiotensin converting enzyme inhibition (ACEi; ramipril 10 mg/day) as a positive control, on renal and systemic hemodynamics, and on RAAS activity (n = 15). RESULTS: Mean (SEM) Glomerular filtration rate (101 (5) mL/min/1.73m2) remained unaffected by DRI or ACEi. Effective renal plasma flow (ERPF; 301 (14) mL/min/1.73m2) was increased in response to DRI (320 (14) mL/min/1.73m2, P = 0.012) and ACEi (317 (15) mL/min/1.73m2, P = 0.045). Filtration fraction (FF; 34 (0.8)%) was reduced by DRI only (32 (0.7)%, P = 0.044). Mean arterial pressure (109 (2) mmHg) was reduced by DRI (101 (2) mmHg, P = 0.008) and ACEi (103 (3) mmHg, P = 0.037). RAAS activity was reduced by DRI and ACEi. Albuminuria (20 [9-42] mg/d) was reduced by DRI only (12 [5-28] mg/d, P = 0.030). CONCLUSIONS: In men with weight excess and hypertension, DRI and ACEi improved renal and systemic hemodynamics. Both DRI and ACEi reduced RAAS activity. Thus, DRI provides effective treatment in weight excess and hypertension. TRIAL REGISTRATION: Dutch trial register, registration number: 2532 www.trialregister.nl.

Mechanism of hyperthyroidism-induced renal hypertrophy in rats.

It is well known that renal hypertrophy is induced by hyperthyroidism; however, the mechanism is not fully understood. We recently reported that cardiac hypertrophy in hyperthyroidism is mediated by enhanced cardiac expression of renin mRNA. The present study addresses the hypothesis that renal hypertrophy in hyperthyroidism is mediated by amplification of renal expression of renin mRNA. Twenty Sprague-Dawley rats were divided into control (n=5) and hyperthyroid groups by daily intraperitoneal injections of saline vehicle or thyroxine. The hyperthyroid group was subdivided further into hyperthyroid-vehicle (n=5), hyperthyroid-losartan (n=5), and hyperthyroid-nicardipine (n=5) groups by daily intraperitoneal injections of saline vehicle, losartan, or nicardipine. All rats were killed at 4 weeks, and the blood and kidneys were collected. The kidney-to-body weight ratio increased in the hyperthyroid groups (+34%). Radioimmunoassays and reverse transcriptase-polymerase chain reaction revealed increased renal renin (+91%) and angiotensin II (+65%) levels and enhanced renal renin mRNA expression (+113%) in the hyperthyroid groups. Losartan and nicardipine decreased systolic blood pressure to the same extent, but only losartan caused regression of thyroxine-induced renal hypertrophy. These results suggest that thyroid hormone activates the intrarenal renin-angiotensin system via enhancement of renal renin mRNA expression, which then leads to renal hypertrophy.

Local renin-angiotensin system contributes to hyperthyroidism-induced cardiac hypertrophy.

We have reported previously that thyroid hormone activates the circulating and tissue renin-angiotensin systems without involving the sympathetic nervous system, which contributes to cardiac hypertrophy in hyperthyroidism. This study examined whether the circulating or tissue renin-angiotensin system plays the principal role in hyperthyroidism-induced cardiac hypertrophy. The circulating renin-angiotensin system in Sprague-Dawley rats was fixed by chronic angiotensin II infusion (40 ng/min, 28 days) via mini-osmotic pumps. Daily i.p. injection of thyroxine (0.1 mg/kg per day, 28 days) was used to mimic hyperthyroidism. Serum free tri-iodothyronine, plasma renin activity, plasma angiotensin II, cardiac renin and cardiac angiotensin II were measured with RIAs. The cardiac expression of renin mRNA was evaluated by semiquantitative reverse transcriptase-polymerase chain reaction. Plasma renin activity and plasma angiotensin II were kept constant in the angiotensin II and angiotensin II+thyroxine groups (0.12+/-0.03 and 0.15+/-0.03 microgram/h per liter, 126+/-5 and 130+/-5 ng/l respectively) (means+/-s.e.m.). Despite stabilization of the circulating renin-angiotensin system, thyroid hormone induced cardiac hypertrophy (5.0+/-0.5 vs 3.5+/-0.1 mg/g) in conjunction with the increases in cardiac expression of renin mRNA, cardiac renin and cardiac angiotensin II (74+/-2 vs 48+/-2%, 6.5+/-0.8 vs 3.8+/-0.4 ng/h per g, 231+/-30 vs 149+/-2 pg/g respectively). These results indicate that the local renin-angiotensin system plays the primary role in the development of hyperthyroidism-induced cardiac hypertrophy.

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with Arthrobacter ureafaciens.

Enzymic hydrolysis of di-D-fructofuranose 1, 2'; 2, 3' dianhydride with the bacteria Arthrobacter ureafaciens was studied to elucidate its mechanism. Hydrolysis of the difructose dianhydride to D-fructose, which did not occur with yeast invertase [EC 3.2.1.26], was found to occur on incubation with an enzyme preparation from an autolysate of the above bacteria. However, incubation with enzyme which had been treated at 60 degrees for 30 min yielded an intermediate hydrolysis product. The product isolated was found to be inulobiose and to be hydrolyzed to D-fructose by the original enzyme, as well as by yeast invertase. It was thus shown that the hydrolysis of the difructose dianhydride to D-fructose with the crude enzyme took place not in a single step but in two separate steps at 2, 3' and 1, 2' linkages. It was not determined whether the entire process is mediated by one and the same beta-fructofuranosidase or by different enzymes.

Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections.

The effects of hydrostatic pressure on subcellular structures, particularly the nucleus, of Saccharomyces cerevisiae were investigated by immunoelectron microscopy. Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature. Frozen thin sections of the cells revealed that spindle pole bodies disappeared at 100 MPa. At 150 MPa, the deposition of gold particles for anti alpha-tubulin was noticed in the nucleus, although the filamentous structure of microtubules was lost. At 200 MPa, fewer gold particles were scattered in the nucleus and the nuclear membrane in several portions was also observed to be open at 300 MPa. These results show that elements of the nuclear division apparatus were susceptible to pressure stress, particularly spindle pole bodies and microtubules. The damage to spindle pole bodies, microtubules, and nuclear membrane caused by pressure stress was followed by the inhibition of nuclear division. After the release of pressure, the spindle pole bodies and microtubules of pressurized cells at below 200 MPa regained their normal appearance at 24 h.

Pituitary, hypothalamic and pancreatic 7B2 concentrations in rats with streptozotocin-induced diabetes and spontaneously diabetic mice.

The pituitary protein, 7B2 has been demonstrated in the pancreas and is known to be present in very high concentrations in pancreatic endocrine tumors. To investigate whether any changes in 7B2 concentrations might be present in the pancreases affected by different types of diabetes and whether the diabetic state itself might affect pituitary and hypothalamic 7B2 concentrations, streptozotocin (STZ)-treated rats and mice with natural-onset diabetes (obese hyperglycemic, or ob/ob, mice and non-obese diabetic, or NOD, mice) were employed. A significant reduction in pancreatic 7B2 concentrations was found in STZ-treated rats. The pancreatic 7B2 concentration was significantly high in ob/ob mice (p less than 0.05, versus the concentration in their lean littermates, and the decrease observed in older NOD mice, appeared to parallel their insulin reserve. Pituitary and hypothalamic 7B2 concentrations were similar in STZ-treated and control rats. A reduction in pituitary and hypothalamic 7B2 concentrations was observed in older NOD mice (both p less than 0.01 versus respective values in younger mice). In ob/ob mice, a significant reduction was also found in hypothalamic 7B2 concentration (p less than 0.01 versus that in control mice). Gel permeation chromatography showed that 7B2 immunoreactivity comprised two molecular components, and the relative proportion in the pancreas differed from that in other tissues. In the pancreas, a smaller molecular component was predominant (elution coefficient, 0.62).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma and urine concentrations of atrial natriuretic peptide in patients with diabetes mellitus.

To examine whether plasma and urine concentrations of human atrial natriuretic peptide (hANP) are altered in patients with diabetes mellitus (DM), plasma and urine hANP concentrations were evaluated in 86 patients with diabetes mellitus using an extraction procedure. The mean recovery rate of extraction was 71.8 +/- 0.6% (mean +/- SEM). The major immunoreactive component of hANP in extracted plasma and urine appeared to be identical to synthetic alpha hANP as judged by reverse-phase high-performance liquid chromatography (HPLC). The patients were divided into three groups according to their renal complications. The patients in group 1 had no apparent abnormality in serum creatinine, serum or urine beta 2-microglobulin (beta 2-MG), or urine N-acetyl-beta-D-glucosaminidase (NAG); those in group 2 showed either beta 2-MG or NAG abnormality but no creatinine abnormality. The patients in group 3 were though to have an established diabetic nephropathy and showed a serum creatinine increase. Plasma ANP concentrations in groups 1, 2, and 3 were 10.7 +/- 2.1, 19.9 +/- 5.6, and 39.2 +/- 9.9 fmol/ml, respectively. These values in groups 2 and 3 were significantly higher than the control values (p less than 0.05 or p less than 0.01 versus 6.2 +/- 0.7 fmol/ml). Urine ANP concentrations in group 1 were also within normal range, though those in groups 2 and 3 markedly increased in comparison with normal values.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.

Simultaneous quantitative determination of 3H, 14C, 32P, 35S, 51Cr, and 125I on the surface of a paper disc using combustion.

A paper disc that retained 3H, 14C, 32P, 35S, 51Cr, and 125I on its surface was combusted in a flow-type combustion flask filled with oxygen. The combustion products containing five radionuclides, except 14CO2, were collected by washing the inner wall of the flask with 0.5-M hydrogen peroxide containing excess NaHSO3. The 14CO2 that remained in the gas phase of the flask was introduced into another flask for absorption into an organic base solution. By determining the radioactivity of both the solutions containing the respective radionuclides by means of NaI(Tl) and liquid scintillation counters, the six radionuclides in the solutions were quantified. The detection limit of surface contamination with this method was compared with the limit specified by Japanese law for the surface contamination of controlled areas.

Pretreatment of animal wastes containing radioisotopes with dry-distillation.

Mice that had been radiolabeled with one of seven isotopes by intraperitoneal or subcutaneous administration were dry-distilled at 800 degrees C for 10 min, and the fate of the radionuclide was examined. The radionuclides administered were 3H, 14C, 32P, 35S, 45Ca, 51Cr, and 125I, all of which are commonly used in animal experiments. After dry-distillation, 95% of 3H was found in the distillate (condensate), and 80% and 16% of 14C were recovered from exhaust gas and residual solids, respectively. Only between 10 and 20% of 35S was found in distillate. The remainder of the residual 35S was recovered from the inner wall of the exhaust vent of the dry-distillation equipment. About 24% of 125I was also recovered from the inner wall of this same exhaust vent, and the residual radioactive materials were recovered from the residual solids. On the other hand, 32P, 45Ca, and 51Cr were recovered entirely from the residual solids. The weight of the animals decreased to about 10%, and the volume to about 20% (mice and rats) to 40% (rabbits) after dry-distillation. The residual solids of animal wastes generated by dry-distillation were odorless and chemically and biologically stable. Dry-distillation has many merits as a pretreatment for the disposal of animal wastes containing radioactive material because of the easy handling, storage, and transportation of the residual solids.

[An experimental study on development of gallstone disease after curative gastrectomy for cancer--postoperative alteration of the composition of canine gallbladder bile after subtotal gastrectomy with truncal vagotomy].

In connection with gallstone (GS) formation after gastrectomy for cancer, we examined alteration of the composition of gallbladder bile after subtotal gastrectomy with truncal vagotomy. Of 11 mongrel dogs with cholecystostomy, 3 underwent B-I and 4 B-II gastrectomy. Four dogs without gastrectomy were served as controls. Bile was collected for 12 months. Bile acids were quantified by GLC. The bile was cultured for bacteriology. TBA did not significantly differ among the three groups of the dogs. In the two gastrectomized groups, CDA (a secondary bile acid) kept a higher level and CA (a primary bile acid) maintained a lower concentration compared with those in the controls. A remarkable increase of non-conjugated bile acids was recognized in both gastrectomized groups. Lithogenicity was low for all of the 3 groups. All but 1 control dog incurred bile infection that persisted long. Black pigment stones containing calcium bilirubinate developed in 1 of the 3 B-I and 2 of the 4 B-II dogs but none in the controls. The bile infection seemed to be involved in the development of GS. As the alteration of bile composition and GS occurred solely in the gastrectomized dogs, subtotal gastrectomy with truncal vagotomy might precipitate the GS formation.

[Immunoreactive 7B2 concentrations in plasma and cerebrospinal fluid in pathophysiological conditions and the responses to oral glucose load, intravenous LH-RH, TRH and arginine infusion].

The peptide, 7B2, originally isolated from pituitary, has been shown to be present in endocrine tumors of high concentrations in pancreatic islet tumors. Plasma from most of these patients showed very high 7B2 immunoreactivity (IR-7B2) though there is a lack of knowledge concerning physiological and pathological changes in plasma IR-7B2 levels in other conditions. To assess whether or not there is any alteration in circulating IR-7B2 levels due to age, sex or any specific condition, plasma levels of IR-7B2 were measured in the fasting state in 106 healthy subjects aged 3 months to 91 years, 101 diabetics, 28 patients with hyperthyroidism. 7 patients with primary hypothyroidism, 13 patients with liver cirrhosis, 43 patients with chronic renal failure, 35 patients with cerebral vascular accident, and 26 pregnant subjects. Twenty-four cord bloods were also included. The responses of circulating IR-7B2 to oral glucose, intravenous arginine infusion, volus thyrotropin (TRH) or volus luteinizing hormone-releasing hormone (LH-RH) injection were also evaluated. Particularly high IR-7B2 levels were found to exist in cord blood. Postnatally the concentrations decreased gradually with age to adult values (15.6 +/- 2.9pmol/liter (mean +/- SE) in 20's-60's), though plasma IR-7B2 levels again increased significantly in over 70's (37.1 +/- 3.2pmol/liter; P less than 0.01). There was no significant difference in plasma 7B2 levels in either sex. Among the pathological conditions studied, significantly high IR-7B2 levels were observed in patients with chronic renal failure (175.1 +/- 35.9pmol/liter). Some of the pregnant patients in their third trimester also showed high plasma IR-7B2 levels. A small but significant rise in plasma IR-7B2 was observed after a glucose load in control subjects and diabetics. Intravenous LH-RH exerted a rise in plasma 7B2 concentrations though arginine and TRH showed no significant effect on plasma IR-7B2 concentrations. Compared with the plasma concentrations, ten to fifty-fold high levels of IR-7B2 were observed in cerebrospinal fluid (CSF) from patients with cerebrovascular accidents or multiple sclerosis. These results suggested that the kidney plays a major role in 7B2 degradation and that LH-RH simulates IR-7B2 release from the pituitary gland. Whether reduced clearance or increased production was responsible for the IR-7B2 elevation in subjects under 10 years or over 70 years requires investigation. Furthermore, high levels of IR-7B2 in CSF might indicate its specific role for the central nervous system.

Estimation of inter-electrode distance for body surface mapping: application of Fourier analysis to potential distribution due to the epicardial breakthrough.

For the purpose of determining an appropriate distance of lead points for constructing body surface maps, Fourier analysis was performed on potential distribution reflecting the epicardial breakthrough, and inter-electrode distance necessary for sampling of harmonics contributing to the body surface potential distribution was determined by application of sampling theorem. Potential distribution was simultaneously recorded with an interval of 4 mm for 250 msec along the vertical (head to foot) and horizontal (right to left) line crossing a second-minimum appearing on the chest surface of a healthy adult, and the data obtained from 9.6 cm along the respective lines underwent Fourier transform. Relative contribution of the second harmonics to the original wave forms increased with time lapse after QRS initiation, in accordance with reductions of the first harmonics component, and attained the maximum (32.5% of total power spectrum without d-c component) at the instant of occurrence of the second-minimum. A similar tendency was observed among higher harmonics, but their contribution was comparatively low (within 10%). Inter-electrode distance determined by sampling theorem was 4.8 cm for the first harmonics and 2.4 cm for the second harmonics. In conclusion it is proposed that placement of electrodes with an inter-electrode distance of 2.4 cm is necessary for acquiring clinically important data on the epicardial breakthrough.

New magnetooptic head with a built-in generator for a bias magnetic field.

A high performance magnetooptic head, which generates a bias magnetic field itself, has been developed. It has a moving magnet actuator to drive an objective lens. The moving magnets have two functions. One is to apply a constant magnetic field on the recording medium. The other is to comprise a magnetic circuit for focusing and tracking control. This magnetooptic head is suitable for use in the two-head erase and write method, in which it is not necessary to change the polarity of the bias magnetic field. Moreover, it enables designing a small-sized magnetooptic disk drive, because a bias magnetic field generator is built into the optical head.

Characterization of intracellular Ca(2+) increase in response to progesterone and cyclic nucleotides in mouse spermatozoa.

Rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca(2+) imaging of the head of individual mouse sperm. Progesterone induced a [Ca(2+)](i) rise in a dose-dependent manner (4-40 microM), primarily in the postacrosomal region. For 20-microM progesterone, Ca(2+) responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1-1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2-4 h as compared with transient responses. 8-Bromo-cGMP (0.3-3 mM) induced a [Ca(2+)](i) rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca(2+) responses (approximately 1 min; 220 nM), independently of the preincubation time. In Ca(2+)-free medium, most sperm showed no Ca(2+) response to progesterone and 8-bromo-cGMP. Pimozide, a Ca(2+) channel blocker, completely blocked prolonged responses and partially inhibited transient responses. These results suggest that progesterone activates at least two distinct Ca(2+) influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response. A cyclic nucleotide-mediated process could participate in the progesterone-induced [Ca(2+)](i) rise.

Bacterial plasmids in antarctic natural microbial assemblages.

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.