Search

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

MPIase is the first known glycolipid that is essential for membrane protein integration in the inner membrane of E. coli. Since the amount of natural MPIase available for analysis is limited and it contains structural heterogeneity, precisely designed synthetic derivatives are promising tools for further elucidation of its membrane protein integration mechanism. Thus, we synthesized the minimal unit of MPIase, a trisaccharyl pyrophospholipid termed mini-MPIase-3, and its derivatives. Integration assays revealed that the chemically synthesized trisaccharyl pyrophospholipid possesses significant activity, indicating that it includes the essential structure for membrane integration. Structure-activity relationship studies demonstrated that the number of trisaccharide units and the 6- O-acetyl group on N-acetylglucosamine contribute to efficient integration. Furthermore, anchoring in the membrane by a lipid moiety was essential for the integration. However, the addition of phosphorylated glycans devoid of the lipid moiety in the assay solution modulated the integration activity of MPIase embedded in liposomes, suggesting an interaction between phosphorylated glycans and substrate proteins in aqueous solutions. The prevention of protein aggregation required the 6- O-acetyl group on N-acetylglucosamine, a phosphate group at the reducing end of the glycan, and a long glycan chain. Taken together, we verified the mechanism of the initial step of the translocon-independent pathway in which a membrane protein is captured by a glycan of MPIase, which maintains its structure to be competent for integration, and then MPIase integrates it into the membrane by hydrophobic interactions with membrane lipids.

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid‐state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three‐dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two‐dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander‐specific protein Prod1 assists cell adhesion in the limb regeneration process.

Advances in peptide biochemistry and mass spectrometry as well as in peptide synthesis and NMR have permitted the isolation, identification and structure determination of numerous products from arachnid venoms, previously unexplored due to technical limitations. The chemical composition in arachnid venoms is diverse ranging from low molecular weight organic compounds such acylpolyamines to complex disulfide-rich peptides. A special group of spider and scorpion peptides are those amphipathic and positively charged structures with cytolytic properties. Their secondary structure is mostly α-helical, and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane of such cells. The mode of action and insertion of antimicrobial peptides from arachnid venoms represent an interesting source of natural molecules for clinical research. In this review, solid-state NMR studies to examine them will be described.

Lipid rafts are cholesterol (Chol)-rich microdomains floating in a sea of lipid bilayers. Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM) rather than with glycerophospholipids, and this putative SM-Chol interaction is generally recognized as a requirement for raft formation. However, the presence of the specific interaction is still controversial, primarily because of the lack of useful molecular probes for scrutinizing this interaction. Recently, we reported that the dynamic properties of 6-F-Chol in DMPC bilayers are similar to those of unmodified Chol. Hence, in the present study, we first compared the roles of 6-F-Chol and Chol in SM bilayers through detergent insolubility, fluorescence polarization, and (2)H NMR experiments. The results demonstrated that 6-F-Chol and Chol behave similarly in SM bilayers, whereas, in SM-DOPC membranes, 6-F-Chol is less effective in domain formation. Then, we analyzed the molecular orientation of 6-F-Chol in SM bilayers using solid-state NMR, and found that the dynamics and orientation of 6-F-Chol in SM bilayers are almost identical to those in DMPC bilayers. This supports the notion of the lack of a putative specific interaction between SM and Chol. Thus, this study demonstrates the utility of 6-F-Chol as a molecular probe for understanding molecular recognition in lipid rafts.

The molecular behavior and interaction of Re-type lipopolysaccharide (ReLPS) and phospholipids were investigated in two different types of model membrane systems, a pure phospholipid membrane consisting of 1,2-dielaidoyl-snglycero-3-phosphoethanolamine (DEPE) and a raft-forming membrane composed of equimolar DEPE, sphingomyelin (SM), and cholesterol (Chol) by solid-state NMR spectroscopy. A remarkable influence of ReLPS on the property of lipid bilayer was found by analyzing the13C-NMR spectra. Namely, while both liquid-ordered (Lo) and liquid-disordered (Ld) phases co-exist in DEPE/SM/Chol, only the Lophase is present in DEPE/SM/Chol/ReLPS. This clearly indicates that ReLPS induces expansion of the raft area in the raft-forming membrane. The1H spin-lattice relaxation times in the rotating frame T1ρHin the two different membranes, DEPE/ReLPS and DEPE/SM/Chol/ReLPS, indicate that the motion of DEPE is affected by the presence of ReLPS, Chol, and SM, and much faster than that of ReLPS in both membranes. The ReLPS in the raft-forming membrane, in particular, accelerated the movement of DEPE. Thus, this study shows the possibility that LPS induces the expansion of raft region and the rapid motion of the raft-forming membranes to favor molecular interactions in the animal cell membrane during innate immune recognition.

Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state 31P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-α-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and (31)P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (DeltaH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were -18.1 kcal/mol and -15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K(p)) was found to be 3.9x10(3) M(-1). (31)P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. (31)P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, (31)P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.

The complete three-dimensional (3D) structure of a glycylisoleucine (Gly-Ile) molecule was determined by individually measuring six dihedral angles with a frequency-selective homonuclear dipolar recoupling method, R2TR (rotational resonance in the tilted rotating frame), using a powder sample of diluted uniformly 13,15-labeled Gly-Ile. Each dihedral angle was obtained by recoupling a dipolar interaction between three or four bonds distant spins concerned or observing a dipolar correlation 2D powder pattern. The 3D structure of a Gly-Ile molecule was also determined by X-ray crystallography, and a good agreement with the NMR result was obtained. The results demonstrate that the R2TR method in a uniformly labeled powder sample can provide the 3D structure without the need to prepare a lot of selectively labeled samples.

A stable HL60 subline having the potential for monocytic differentiation was established by use of GM-CSF. HL60, a human promyelocytic cell line, has frequently been employed for research in the fields of monocytic differentiation and atherosclerosis because of its potential to differentiate into either granulocytes or monocytes. However, HL60 are frequently seen to change their phenotype during long-term culture. To date, many sublines or variants of HL60 cells have been established. However, most of them display diminished or complete loss of activities that characterize parental cells. The present study was conducted to establish a stable HL60 subline with the potential for monocytic differentiation. Firstly, a single HL60 cell was isolated by limiting dilution, and was successfully proliferated by incubation with GM-CSF. Secondly, from this population, cells were selected that had the ability to generate superoxide after VD-induced monocytic differentiation. Cells obtained in this manner (designated HL60/DU-1) exhibited expression of CD14 and CD11b and suppression of CD3 expression after monocytic differentiation. NBT positivity showed a consistent level of over 971% after a 6-day challenge with VD throughout the experimental period of 12 months. HL60/DU-1 cells, which were cryopreserved in liquid nitrogen for 6 months, thawed and re-cultured, exhibited over 97% NBT positivity. Carvedilol and probucol, which exhibit antioxidative activity, inhibited superoxide release from the differentiated HL60/DU-1 cells. HL60/DU-1 cell line is a promising model for the study of monocytic differentiation and the effects of oxygen radicals.

Lipopolysaccharide (LPS) is a key molecule that triggers the immediate response of the innate immune system in higher animals against invading Gram-negative microbes. Solid-state NMR spectroscopy revealed how ReLPS, a deep rough mutant LPS of Escherichia coli, effects the morphology, phase state and molecular motion of the membrane. ReLPS exerted a substantial influence on the phase state of raft-forming membranes comprising equimolar 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), sphingomyelin (SM) and cholesterol (Chol), as observed using 13C NMR. Both liquid-ordered (Lo) and liquid-disordered (Ld) phases coexist in the DEPE/SM/Chol membrane, whereas the Ld phase disappeared on addition of LPS. This clearly indicates that ReLPS has a capability to expand the “raft” area in raft-forming membranes. NMR relaxation times indicate that the effects of ReLPS incorporation on the motions of the DEPE and DEPE/SM/Chol membranes are opposite. In the DEPE membrane, lipid dynamics were slower than that in the absence of ReLPS. By contrast, in the raft-forming membrane, ReLPS reduced the packing effect of Chol and led to faster lipid motions. Thus, in addition to its role as ligand during the innate immune recognition, LPS may act autocatalytically in animal cell membranes by inducing the expansion of raft regions and rapid motions in membranes to facilitate molecular interactions.

An application of the R2TR method (1995, Chem. Phys. Lett. 232, 424) to selective homonuclear polarization transfer under magic angle spinning is proposed. It is shown that, for a spinning speed fast enough to remove the maximum homonuclear dipolar coupling constant ω D involved, the flip-flop and flop-flop mechanisms are suitable for recoupling the spins with a chemical shift difference larger than ω D and a difference comparable to or smaller than ω D , respectively. It is also shown that, for fast polarization transfer, the off-resonance frequencies should be much higher than the RF intensity in the flip-flop condition, while for the flop-flop condition, the off-resonance frequencies should be much lower than the RF intensity. Some one- and two-dimensional experiments are proposed by utilizing the capability of the R2TR method to abruptly switch on and off the recoupling condition, and are demonstrated for triply 13 C-enriched l -alanine. The mixing time required for population transfer was found to be ca. 0.5 ms for the methine and methyl 13 C spins separated by 1.5 A and ca. 5 ms for the methyl and the carboxyl carbons separated by 2.5 A. The experimental results and theoretical simulations show that selective polarization transfer is achieved when the difference in the isotropic chemical shifts between the relevant pair of spins and a neighboring spin is more than 1000 Hz.

We propose a new solid-state NMR technique under magic-angle spinning to recouple the dipolar interaction for a particular pair of homonuclear spins. In this method, a weak radio-frequency field is applied during the mixing time of the exchange NMR, and its frequency and intensity are chosen to realize rotational resonance for a particular pair of spins in the tilted rotating frame. The selective recoupling is demonstrated for the dipolar interaction between the CH and CH 3 carbons in triply 13 C-enriched l -alanine, whose chemical shift difference is too small to apply the conventional rotational resonance method.

We examined the protective effects of medical castration by means of gonadotropin-releasing hormone analogue (GnRHA) on the toxic effects of cisplatin in rats. Twelve days after a s.c. injection of a slowly-releasable form of leuprolide acetate (GnRHASR), rats were injected i.p. with cisplatin daily (3 mg/kg body weight (BW) for males and 4 mg/kg BW for females) for four days and sacrificed 24 h after the last injection. The doses caused acute tubular necrosis and gastrointestinal (GI) symptoms, i.e., diarrhea and fluid retention and bleeding in GI tract. GnRHASR pretreatment reduced serum urea nitrogen (SUN) and serum creatinine (SCre) increase and the incidence of GI symptoms. Histological analysis showed that rats pretreated with GnRHASR had noticeably less kidney damage. GnRHA thus demonstrated its ability to protect the kidneys and GI tract against cisplatin toxicity in both male and female rats. This finding suggests a potential clinical application of GnRHA in antineoplastic chemotherapy.

A small subgroup of primary aldosteronism due to aldosteronoma, named aldosterone-producing renin-responsive adenoma (AP-RA), has been reported to masquerade as idiopathic hyperaldosteronism (IHA) because of the responsiveness of the plasma aldosterone concentration (PAC) to upright posture (UP). We found two patients with AP-RA in 19 patients with aldosteronoma who were examined by UP stimulation and were treated surgically. In 17 patients with typical aldosterone-producing adenoma (APA), PAC decreased or increased only slightly (less than 200% of the basal level); in contrast, it increased to over 300% of the basal level in two patients with AP-RA. The two groups were comparatively studied as to their hormonal levels, adrenal computed tomography (CT) scan and histological findings in order to clarify the characteristics of AP-RA. Basal PAC was within the normal range (11.1 and 13.0 ng/dl) in AP-RA but in APA it ranged from 14.8 to 58.1 ng/dl with a mean of 32.3 +/- 2.7 ng/dl. The diameters of the adenoma in AP-RA were apparently smaller (6 and 9 mm) than those in APA ranged from 10 to 25 mm with a mean of 15.5 +/- 1.1 mm. After a contrast medium was injected at CT scan, the density of the normal adrenal gland adjacent to the adenoma increased but that of the adenoma did not in APA, making a clear distinction between the adenoma and the gland. On the other hand, the density of the adenoma and gland increased to almost the same degree in AP-RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Nineteen patients with primary aldosteronism due to surgically confirmed aldosterone-producing adenoma (APA) were examined to evaluate the response of aldosterone to upright posture and angiotensin II infusion. Upright posture reportedly decreases the plasma aldosterone concentration (PAC) in APA but raises it in idiopathic hyperaldosteronism. However, our findings showed the opposite result, in that the upright posture did not change or raised PAC in 15 of 19 cases (79%). Angiotensin II was infused i.v. at doses from 0.5-2 ng/min.kg body weight in six patients in whom the upright posture raised PAC, but did not raise PAC in all cases. This result supports the assumption that APA is functionally insensitive to angiotensin II. A concomitant rise of ACTH, pretreatment with calcium channel blockade, and other modulating factors may be involved in this PAC rise. Whatever the reason, such a high frequency of patients with increased PAC in APA raises some question about the clinical value of the upright posture test. We believe, then, there is reason to check any interpretation concerning increased PAC in the case of the upright posture test in distinguishing between APA and idiopathic hyperaldosteronism.

We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile , a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile ( n = 4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N -3-oxododecanoyl l -homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 μM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile .

2850, 2856

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.

6-F-cholesterol was reported to exhibit biological and interfacial properties similar to unmodified cholesterol. We have also found that 6-F-cholesterol mimicked the cholesterol activity observed in the systems of amphotericin B and lipid rafts. However, to use 6-F-cholesterol as a molecular probe to explore molecular recognition in membranes, it is indispensable to have detailed knowledge of the dynamic and orientation properties of the molecule in membrane environments. In this paper, we present the molecular orientation of 6-F-cholesterol (30 mol %) in dimyristoylphosphatidylcholine (DMPC) bilayers revealed by combined use of 19F chemical shift anisotropy (CSA), 2H NMR, and C-F rotational echo double resonance (REDOR) experiments. The axis of rotation of 6-F-cholesterol was shown to be in a similar direction to that of cholesterol in DMPC bilayers, which is almost parallel to the long axis of the molecular frame. The molecular order parameter of 6-F-cholesterol was determined to be ca. 0.85, which is within the range of reported values of cholesterol. These findings suggest that the dynamic properties of 6-F-cholesterol in DMPC are quite similar to those of unmodified cholesterol; therefore, the introduction of a fluorine atom at C6 has virtually no effect on cholesterol dynamics in membranes. In addition, this study demonstrates the practical utility of theoretical calculations for determining the 19F CSA principal axes, which would be extremely difficult to obtain experimentally. The combined use of quantum calculations and solid-state 19F NMR will make it possible to apply the orientation information of 19F CSA tensors to membrane systems.

Dexamethasone-suppression (DS) adrenal scintigraphy localizes an aldosteronoma, but with false-negative results, i.e. 2 of 19 cases in our study. Our aim was to clarify the clinical meaningfulness of this test. Adrenal iodomethyl-norcholesterol (NP-59) uptake on the adenoma side correlated with the estimated adenoma volume (n = 15, r = 0.843, P less than 0.001). Accordingly, the uptake ratio on the adenoma side to that on the opposite side depended on the adenoma volume (r = 0.683, P less than 0.01). This explains the false-negative results (uptake ratio less than 2) in two cases with small adenomas. The NP-59 uptake correlated weakly with the plasma aldosterone level (r = 0.516, P less than 0.05). This result indicates the low correlation between NP-59 uptake and the ability to secrete aldosterone. NP-59 accumulation in the surgically removed gland was analyzed by autoradiography in six cases where DS scintigraphy was done just before surgery. The density was higher in the adenoma cells than in the adjacent cortical cells in five cases, but the difference was rather small, i.e., within a 2-fold difference in four cases. In one case, almost the same density was observed in both types of cells. Thus, the laterality of NP-59 uptake primarily depends on the adenoma volume although NP-59 uptake somewhat reflects the adenoma's ability to secrete aldosterone or the adenoma cell's activity in accumulating NP-59. Care must be taken in interpreting the findings from DS scintigraphy where the adenoma is small or adrenal uptake is low.

Objective: The effect of nutritional state on lymphocytes in patients with anorexia nervosa (AN) was studied. Method: We studied total lymphocyte count (TLC), lymphocyte subsets, and nutritional markers [body mass index (BMI), insulin-like growth factor-1 (IGF-I)], and serum zinc concentration) in 33 patients with AN and 10 healthy controls. Results: TLC positively correlated with BMI (r = .680, p < .001), IGF-I (r = .609 p < .001), and zinc (r = .589, p < .001). The CD4+ T-lymphocyte (CD4) proportion correlated negatively with BMI (r = −.301, p = .05) and IGF-I (r = −.346, p = .023), counteracting the effect of malnutrition on TLC. However, because this increase in CD4 proportion was weak, patients with very severe malnutrition (indicated by serum zinc less than 40 μg/dL) had critically low CD4 counts of less than 200 cells/μL. Conclusion: Our findings suggest that lymphocyte counts and subset proportion change in an opposite manner in patients with AN, and that decrease in serum zinc levels is nutrition-related. © 2007 by Wiley Periodicals, Inc.

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly3, Gly8, Leu12, Phe17, or Ser18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala4, Gly8, and Ala9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.

In order to determine precise three-dimensional structures of proteins by residual dipolar coupling constants as the major or even exclusive structural constraints, it is essential to use two anisotropic media. In doing so, a reliable and versatile method for estimating the relative orientation of the alignment tensors for the molecules dissolved in different anisotropic media is required. In this communication, we present a new graphical approach for this purpose, which does not require structural information of the target molecules. The correlation map for the two independent data sets of residual dipolar coupling constants, which can be obtained for the molecules in different anisotropic media, strongly depends on the relative orientation of the alignment tensors. We have simulated the correlation maps for all possible combinations of the Euler angles, which transform one alignment tensor to the other, and compared them to the experimental data sets reported for labeled human ubiquitin. This simple graphical method affords a useful starting point for the structural determinations using residual dipolar couplings.

An aldosterone-producing adenoma causes surgically correctable hypertension. Screening tests should be assessed for their accuracy and ability to detect aldosterone-producing adenoma in an appropriate population. This study aims to validate the accuracy and efficacy of the basal plasma aldosterone concentration (picomoles per liter) to PRA (nanograms per liter/sec) ratio and of combined stimulation of PRA by the furosemide and upright posture test in screening for aldosterone-producing adenoma in hypertensives with PRA less than 0.28 ng/liter.sec (1 ng/ml.h). Thirty-five aldosterone-producing adenoma and 79 nonaldosterone-producing adenoma patients were retrospectively selected from among 159 patients examined with the furosemide and upright posture test between 1989 and 1999. Selection criteria were based on blood pressure, PRA, and plasma aldosterone concentration. Diagnosis was based on surgical outcome, computed tomography scans with adrenal scintigraphy, or venous sampling. The accuracy and efficacy of basal (aldosterone/PRA ratio) and dynamic (postfurosemide and upright posture PRA) screening tests were assessed based on test sensitivity, specificity, likelihood ratio, and receiver operating characteristics. At a cut-off value of 3,200, the aldosterone/PRA ratio had a high sensitivity of 1.0 and a low specificity of 0.61. The importance was strengthened by using a multilevel likelihood ratio, i.e. positive (aldosterone/PRA ratio >10,000), negative (aldosterone/PRA ratio

Systemic and nonspecific stress response effects on the cellular defense mechanism were studied in the male rat kidney. Two days after laparotomy-induced surgical stress, rats showed increased serum corticosterone and renal cortical reduced glutathione (GSH). Rats were then injected s.c. with mercuric chloride (HgCl2) to oxidatively injure renal tubuli. Increased serum creatinine levels indicated that laparotomy pretreatment lessened renal damage. To study the effects of the activated pituitary-adrenal axis on renal cortical GSH content and vulnerability to subsequent oxidative injury, rats were injected s.c. with ACTH on two consecutive days. ACTH administration increased both corticosterone and aldosterone. These rats showed increased, dose-dependent renal cortical GSH content, i.e., controls (n=7): 1.25 +/- 0.23 micromol/g tissue, daily dose of 10 microg/100 gBW (n=7): 1.53 +/- 0.24 micromol/g tissue, and daily dose of 40 microg/100 gBW (n=7): 2.31 +/- 0.23 micromol/g tissue. Rats receiving daily doses of 40 microg of ACTH/100 gBW acquired resistance to oxidative injury, indicated by serum creatinine levels: controls (n=6), 22 +/- 4 micromol/L; HgCl2 (n=6), 145 +/- 88 micromol/L; ACTH and HgCl2 (n=6), 37 +/- 11 micromol/L. Morphological evidence indicated that ACTH pretreatment in HgCl2-injected rats prevented renal tissue from inflammatory cell infiltration but not from tubular degeneration. Cellular GSH content of LLC-PK1 cells, porcine renal-tubule-derived culture cells, increased significantly in incubation with dexamethasone or aldosterone, suggesting that adrenal steroids directly stimulate renal cell GSH. We demonstrated that stress or ACTH administration activates the defense mechanism in the kidney via increased GSH. This stress-activatable defense system may therefore indicate a connection between endocrine stress response and the cellular defense mechanism.

OBJECTIVE To elucidate the relationship between the activity of CMV disease and adrenocortical function in patients with AIDS. DESIGN AND PATIENTS CMV retinitis and CMV antigenemia assay (CMV-Ag: numbers of polymorphonuclear leukocytes positive for CMV pp65 antigen per 1.5 x 10(5) cells) are the least invasive and easily accessible examinations to assess the CMV disease activity. All HIV-infected patients with CD4+ lymphocyte counts < 50 x 10(6)/l who were admitted to the Research Hospital of the Institute of the Medical Science (University of Tokyo) between May 1995 to April 1996 were included in this study. METHODS Fundoscopic examination on CMV retinitis and CMV-Ag were chosen as methods to assess CMV activity because of their simplicity. Adrenocortical function was evaluated by basal plasma adrenocorticotropin, plasma cortisol, plasma aldosterone, plasma renin activity, and responses of plasma cortisol and plasma aldosterone to 250 micrograms intravenous cosyntropin [rapid adrenocorticotropin test (RAT)]. RESULTS Thirty patients were enrolled in this study with a maximum CD4+ lymphocyte count of 32 x 10(6)/l. Eleven out of 30 patients showed impaired RAT response (37%). Fourteen out of 30 patients had CMV retinitis. A significant correlation was found between the presence of CMV retinitis and subnormal cortisol response (P < 0.005). Sixteen out of the 30 patients were CMV-Ag-positive. A significant correlation was found between CMV-Ag positivity and subnormal cortisol response to RAT (P < 0.005). CMV-Ag levels in the patients with subnormal cortisol response to RAT were significantly higher than those with normal response (P < 0.001). Importantly, five patients with subnormal cortisol response but not overt adrenal insufficiency at the time of RAT developed overt disease shortly afterwards. Autopsy was performed in six patients with subnormal cortisol response and showed multiple inclusion bodies indicative of CMV adrenitis. CONCLUSION The adrenal gland is most frequently affected by CMV in AIDS patients. Our result suggests that CMV retinitis or CMV-Ag positivity independently serve as an indication of possible adrenal dysfunction.

Hypertension and norepinephrine hypersecretion in a 59-year-old woman suffering from malignant pheochromocytoma with multiple metastases were appropriately controlled with alpha- and beta- blockers, and alpha-methyltyrosine (alpha-MT), a catecholamine-synthesis inhibitor. Metastasized vertebrae were treated with external radiation to relieve pain, but this treatment had to be interrupted at a total dose of 20 Gy because the patient suffered acutely exacerbated hypertension (200/110 mmHg), tachycardia (160 beats/min) and a low-grade fever. Simultaneously her serum levels of LDH, potassium, urea nitrogen, creatinine, white blood cell count, CRP and norepinephrine were significantly increased, suggesting that this episode was due to radiation-induced tissue destruction and the leakage of catecholamines and possibly interleukin-6, a cytokine mediating inflammation which is reportedly present in pheochromocytoma. The marked hypertension was controlled by continuous i.v. administration of phentolamine and propranolol. Although radiation therapy effectively relieves pain due to neoplasmic metastasis to the bone, physicians should be aware that life-threatening complications such as the above occur in malignant pheochromocytoma. Sufficient pretreatment with adrenergic blocking agents and/or alpha-MT and careful monitoring of the patient's general condition during radiation therapy, even at a low dose, are highly recommended.

The present study surveyed 69 patients with aldosteronoma to study the clinical implications of renal cysts demonstrated in computed tomography. Patients who had cysts (n = 16, 23.2%) were older and had a longer duration of hypertension and more severe hypokalemia than those without cysts (n = 53). Patients with cysts therefore had longer-term, more severe hypokalemia than those without cysts. Endogeneous creatinine clearance (Ccr), measured in 61 patients, was significantly lower in patients with cysts (58.4 +/- 7.1 ml/min, n = 16) than in those without cysts (77.3 +/- 7.1 ml/min, n = 45, P = 0.0039). This significant difference was observed even after adjusting for covariables (age, duration of hypertension, and serum potassium) between the two groups by analysis of covariance (ANCOVA). No significant difference was observed in gender, blood pressure, serum creatinine, plasma aldosterone, or PRA. Age, serum potassium levels, and systolic and diastolic blood pressure were the significant determinants in predicting Ccr in a backward stepwise multiple regression analysis (r = 0.505, n = 61, P = 0.0025). Cysts were graded into four classes on the basis of number and size. Cyst grading correlated negatively with Ccr at a Spearman rank correlation (rho = -0.33, n = 61, P = 0.0103). The incidence of chronic renal failure was significantly higher in patients with cysts (18.8%) than in patients without (0%) in a Fischer's exact probability test (P = 0.0107). Thus, both renal cysts and dysfunction arose and/or developed from common roots, i.e., the duration and severity of hypokalemia, in primary aldosteronism. In addition, we surveyed 27 patients with pheochromocytoma. Patients with renal cysts (n = 8) had a significantly longer duration of hypertension than those without cysts. No significant difference was observed in Ccr between patients with and those without cysts. Thus, a significant link between renal cysts and Ccr was a specific feature of primary aldosteronism, but not of pheochromocytoma. In summary, the renal cysts in primary aldosteronism should be recognized as a significant complication representing the extent of renal injury and dysfunction.

Cysteamine, a specific somatostatin depleter, was given to male rats to clarify its role in relation to the renin-angiotensin-aldosterone (RAA) axis and glomerulosa cell growth. Rats received seven daily sc injections of cysteamine at doses of 50 or 150 mg/kg body weight (BW). Their adrenal weights and whole cortical thickness increased, but zona glomerulosa thickness decreased dose-responsively. Plasma renin activity (PRA) and aldosterone concentration (PAC) decreased. Similar results were observed in rats on a low or high salt diet and receiving daily doses of 150 mg/kg BW of cysteamine. In hypophysectomized rats, however, cysteamine given for seven days at daily doses of 100 mg/kg BW did not change either PRA or PAC. Adrenal weight did not change either too. Our results indicate that cysteamine suppresses the RAA axis and glomerulosa cell growth, probably through pituitary factors.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Some of luteinizing hormone (LH) isoforms can stimulate renal growth. The objective of this study is to determine whether the administration of LH modifies subtotal nephrectomy-induced chronic renal failure. Castrated 3/4-nephrectomized male rats were divided into four groups of seven each and fed a low-protein (6%) diet. Ovine LH with renotropic activity (40 micrograms/day) or vehicle only (control) was given for three weeks or six weeks. Compared with controls, remnant kidney weights (% body weight) in LH-treated rats had increased significantly at three weeks (0.385 +/- 0.019 vs 0.443 +/- 0.052, P less than 0.02), but not at six weeks (0.281 +/- 0.004 vs 0.272 +/- 0.013). 24 h creatinine clearance (ml/day/100 g body weight) increased significantly both by three weeks (242 +/- 58 vs 301 +/- 36, P less than 0.05), and six weeks (323 +/- 55 vs 395 +/- 10, P less than 0.01). Urinary thromboxane B2 excretion increased in LH-treated rats, suggesting that hemodynamic changes may play a role in increasing creatinine clearance. Our results suggest that renotropically active oLH stimulated the glomerular function in castrated rats with reduced renal mass. Further study may clarify its clinical usefulness.

LH is composed of isoforms which exhibit microheterogeneity. We recently demonstrated that a particular ovine or porcine LH preparation (G100-fr.3) stimulates kidney growth. This study was conducted to clarify the physiological role of this renotropic activity and other extragonadal effects of the ovine LH preparation in CD-1 mice. Hypophysectomy caused a significantly greater reduction in relative dry kidney weight (i.e. g/100 g body weight) when compared to adrenalectomy, castration, thyroidectomy, and castration plus thyroidectomy. Supplementation with G100-fr.3 in these animals partially restored not only kidney size but also DNA, RNA and protein content. Treatment with standard LH preparations (NIDDKoLH24 and G3-268DA), as well as PRL, GH, FSH and TSH, failed to reverse the renal atrophy induced by hypophysectomy and castration. Administration of testosterone to castrated hypophysectomized mice increased kidney weight and RNA content, but not renal DNA. The relative dry kidney weight increased significantly at the onset of puberty in intact male mice, but not in castrated males or intact female mice. In addition, human CG increased kidney size in hypophysectomized male mice, but not in castrated hypophysectomized animals. These findings indicate that LH isoforms may regulate kidney growth in the male mouse both directly as a renotropin stimulating hyperplasia and indirectly as a gonadotropin via testicular androgen, producing cellular hypertrophy. It was also noted that G100-fr.3 decreased hepatic weight, DNA, RNA and protein, but produced no significant change in the spleen, heart or adrenal glands in castrated-hypophysectomized mice. Such extragonadal effects of G100-fr.3 were also observed in intact female mice. These results suggest that certain LH isoforms may have extragonadal actions involving the kidney and liver.

A highly sensitive and simple radioimmunoassay for the measurement of 8-arginine vasopressin (AVP) in human plasma has been developed. The dose response curve ranges from 0.025 to 8 pg/tube. This simple extraction technique employing an ODS C18 column recovered 87.1 +/- 10.4 (mean +/- SD)% of AVP in the range of 1-10 pg/ml added to 0.5 ml plasma. Determination of AVP in each fraction of plasma, which was gel-filtrated through a Sephadex G25 (1 X 25 cm), revealed that the fraction of plasma AVP was superimposed on that of authentic AVP, and interference of non-specific substances was completely eliminated by an ODS C18 column. Using the assay, 13 of 16 patients with diabetes insipidus (DI) showed plasma AVP concentrations ranging from 0.03 to 0.21 pg/ml, and the other 3 patients had less than 0.03 pg/ml. The AVP concentrations of DI were clearly distinguished from those obtained in normal subjects (0.30-4.20 pg/ml, n = 65). The within and between assay variability was about 10% each. Plasma AVP concentrations (mean +/- SD) of normal subjects (n = 6) standing, sitting, and supine after an overnight of fluid deprivation were 2.41 +/- 1.15, 1.95 +/- 0.85 and 0.97 +/- 0.48 pg/ml (30 min after) respectively. Plasma AVP concentrations of normal subjects (n = 6) after water load (20 ml/kg wt) were clearly reduced from 1.89 +/- 1.00 (before) to 0.42 +/- 0.21 (standing for 60 min) and also from 0.89 +/- 0.41 to 0.40 +/- 0.22 pg/ml (supine for 60 min).

Kidney slices were obtained from castrated-hypophysectomized male rats treated with a single injection of several different gonadotropin preparations (two ovine LH fractions, one bovine LH fraction, and one hCG fraction) or vehicle, then incubated in a buffer containing [3H]thymidine. Only one of the above, an ovine LH preparation, increased [3H]thymidine incorporation into renal DNA, with a peak occurring 8-10 h after injection and therefore termed renotropin. However, in hypophysectomized rats with intact testes, such an effect was not observed. Furthermore, while testosterone propionate alone did not alter basal incorporation in castrated-hypophysectomized rats, it abolished the incorporation that was stimulated by renotropin. These results suggest that androgens, whether of testicular origin or exogenously administered, suppress the increased incorporation of [3H]thymidine stimulated by renotropin. This antagonistic effect of androgen was also observed with T4, but to a lesser degree. Our findings confirm the presence of renotropin, which could not be attributed to other known pituitary hormones, and suggest that there is a complex interaction between this factor and two other renal growth-promoting hormones, testosterone and T4.

In a primary culture of bovine adrenal zona glomerulosa cells, insulin-like growth factor I (IGF-I)/somatomedin C stimulated DNA synthesis, as measured by [3H] thymidine uptake, at concentrations of 10(-9) and 10(-7) M. IGF-I also prevented ACTH-induced suppression of [3H] thymidine uptake. IGF-I in no way affected aldosterone secretion during short-term exposure to cultured cells, however. Our findings suggest that IGF-I plays an important role in the proliferation of adrenal zona glomerulosa cells.

Corticotropin-releasing factor (CRF)-like activity in two different kinds of ACTH-producing tumors (human ectopic ACTH-producing colonic cancer and rat MtT/F4 tumor) was determined by an in vitro method using isolated normal rat pituitary cells. The ACTH content of the post mortem colonic cancer was 5.5 ng/g w.wt. The ACTH content of the medium of MtT/F4 tumor cells was 153+/-32 pg/10(5) cells. The ACTH content of MtT/F4 tumor cell suspensions was elevated with increasing doses of hypothalamic median eminence extract (HME). The response of MtT/F4 tumor cells to HME was suppressed by 1 mug/ml of dexamethasone. Extracts of colonic cancer, MtT/F4 tumor and HME produced elevation of the ACTH content of the medium of isolated rat pituitary cells. The CRF-like activities of two kinds of tumor extracts in multiple dilutions ran parallel to that of HME. The CRF-like activities were 0.037 HME equiv/mg w.wt in MtT/F4 tumor and 0.052 HME equiv/mg w.wt. in colonic cancer. These results demonstrated that CRF-like activity existed in these two kinds of ACTH-producing tumors.

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct alpha-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10(-3) s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at approximately 30 degrees to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.

Three patients with idiopathic hyperaldosteronism were continuously treated with trilostane, a competitive inhibitor of adrenal 3β-hydroxysteroid dehydrogenase (3β-HSDH) (3 to 4⅔ years). Trilostane, in conjunction with antihypertensive drugs, effectively decreased plasma aldosterone levels and improved hyperaldosteronism symptoms without undesirable side effects. Trilostane continued to be effective even when treatment was continuous. Rapid ACTH testing (iv bolus of 0.25 mg α1–24 ACTH) was done on the day without trilostane after long-term treatment, and plasma levels of aldosterone and cortisol were compared to those obtained during a pre-treatment period. Results suggest that the inhibitory effect of trilostane on steroid biosynthesis rapidly disappears following discontinuance of trilostane administration even after long-term treatment, and that continuous treatment causes no significant or irreversible change in steroid biosynthesis. These results suggest that trilostane is a safe, feasible therapeutic agent for long-term treatment of idiopathic hyperaldosteronism.

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

This study was done to confirm that aldosterone-producing adenomas secrete cortisol in vivo. Plasma cortisol and aldosterone concentrations were measured in samples obtained by selective adrenal-vein sampling in 8 patients with primary aldosteronism due to unilateral adenoma. All cases revealed higher adrenal-vein plasma cortisol concentrations on the adenoma side than the opposite, irrespective of adenoma location. These concentrations correlated significantly with plasma aldosterone concentrations (r = 0.972, P < 0.001) in effluents from the adenoma side, but not from the opposite. Plasma concentrations also correlated significantly with estimated adenoma volume (r = 0.918, P < 0.05). These findings strongly suggest that aldosterone-producing adenomas secrete cortisol in vivo. In a second study, we used metyrapone to test 6 patients with adenomas. Their responsiveness to cortisol and corticotrophin was found to be the same as that in normal subjects, suggesting that adenoma-secreted cortisol did not disturb the relationship between corticotrophin and cortisol. We thus concluded that cortisol is secreted concomitantly with aldosterone from aldosterone-producing adenomas under corticotrophin influence.

We recently demonstrated the renotropic activity in ovine LH isoform(s). In this study we purified porcine (p) LH isoforms and analyzed their structures and bioactivities. Purified pLH preparation (G-100 fraction 3) was dissociated into alpha- and beta-subunits, followed by isolation with reverse phase HPLC. Six components were isolated and analyzed for their amino acid compositions and amino-terminal amino acid sequences. alpha-Subunits were found to have heterogeneous N-terminal sequences, which started at Phe-1, Gly-4, Phe-6, or Thr-7. This N-terminal heterogeneity and the presence of the initial 6 amino acids has not been reported previously to the best of our knowledge. Moreover, the first 10 residues, Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly, were identical with those of the ovine LH alpha subunit. At least 3 different beta-subunits were identified as heterogeneous in their carboxyl-terminal amino acids. The pLH preparation (G-100-fraction 3) was then chromatographed by an isoelectrofocusing (IEF) column. Four IEF fractions were obtained. Their amino acid structures appeared to be identical, as judged by the identical elution profiles on reverse phase HPLC, but their carbohydrate compositions were slightly different, especially in N-acetylgalactosamine in alpha-subunits. Thus, fractionation on IEF depended on the heterogeneity in carbohydrate structures. Each IEF fraction had different potencies in terms of its gonadotropic activity (in vitro) and renotropic activity (in vivo and in vitro). The discrepancy was observed not only between gonadotropic activity and renotropic activity, but also between in vivo and in vitro renotropic activity. This study identified the structural heterogeneity of pLH isoforms and demonstrated their biological heterogeneity. We concluded that the carbohydrate structure of the pLH isoform is important for expressing its biological heterogeneity (gonadotropic and renotropic activity.

A 47 year old woman examined for hypertension (200/100 mmHg) was normokalaemic, and had low plasma renin activity (PRA) (0.1 ng/ml · h) and normal aldosterone levels in both plasma (7–13 ng/dl) and urine (4.7–7.4 μg/day). Computed tomography (CT) and scintiscan indicated an adenoma on the right adrenal gland, which was then removed. The histology of the adenoma and analysis of the aldosterone content were compatible with the criteria for an aldosterone-producing adenoma. Three months after surgery, her hypertension had improved, serum potassium levels had increased slightly, and PRA had normalized. This was an unusual form of primary aldosteronism which showed normal levels of aldosterone in both blood and urine.

We explored the effects of ovine pituitary-derived renotropin on renal DNA synthesis in castrated hypophysectomized mice. Administration of the preparation at a dose of 47 micrograms for 5 days was followed by significant increases in renal DNA (134% of controls), and in kidney dry weight, protein and RNA. A time course study showed that [3H]-thymidine incorporation into renal DNA peaked at 8-10 h after one injection (60 micrograms) 1.9 times higher than in controls. Autoradiographic studies indicated that labeling indices increased significantly in proximal tubules (17 times) and endothelial cells (4 times) in the outer renal medulla of treated mice compared to controls. Nuclear areas in these cells also increased significantly. Our studies demonstrated a time course of new DNA synthesis stimulated by a renotropin and identified renotropin target cells.

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.