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Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in

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.

Inhomogeneous line broadening due to conformational distributions of molecules is one of the troublesome problems in solid-state NMR spectroscopy. The best possible way to avoid it is to crystallize the sample. Here, we present a highly resolved (13)C cross-polarization (CP) magic angle spinning (MAS) NMR spectrum of the highly ordered crystalline 1,2-dimyrystoyl-sn-glycero-3-phosphocholine (DMPC) and completely assigned it using two-dimensional (2D) solid-state NMR spectra, dipolar heteronuclear correlation (HETCOR) spectra, scalar heteronuclear J coupling based chemical shift correlation (MAS-J-HMQC) spectra, and Dipolar Assisted Rotational Resonance (DARR) spectra. A comparison between assigned chemical shift values by solid-state NMR in this study and the calculated chemical shift values for X-ray crystal DMPC structures shows good agreement, indicating that the two isomers in the crystalline DMPC take the same conformation as the X-ray crystal structure. The phase diagram of the low hydration level of DMPC (3 ≤ n(W) ≤ 12) determined by (1)H and (13)C NMR spectra indicates that DMPC takes a crystalline state only in a very narrow region around n(W) = 4 and T < 313 K. These findings provide us with conformational information on crystalline DMPC and the physical properties of DMPC at a low hydration level and can possibly help us obtain a highly resolved solid-state NMR spectrum of microcrystalline membrane-associated protein samples.

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.

Patients with adrenal insufficiency have a life-threatening risk of adrenal crisis, thus preventing adrenal crisis is an important clinical issue. In order to clarify the risk factors for adrenal crisis, the medical records of 137 patients with established adrenal insufficiency were retrospectively investigated. The explanatory variables analyzed were gender, etiology of hypoadrenalism, class of adrenocortical hormone replaced, duration of steroid replacement, age at time of survey, age at time of diagnosis of hypoadrenalism, state of other hormone deficiencies (growth hormone and sex steroids), diabetes insipidus, and mental disorder. Diagnosis of adrenal crisis was based on physical and laboratory findings. Forty (29%) of the 137 patients had at least one episode of adrenal crisis. Based on the Akaike Information Criterion (AIC), steroid replacement therapy of more than 4 yrs' duration was the largest single contributor to the occurrence of an adrenal crisis, followed by mental disorder and sex steroid deficiency. In the subclass of patients with secondary adrenal insufficiency (N = 115), sex steroid deficiency was the greatest risk factor. Patients with untreated hypogonadism had a significantly higher relative risk of 3.70 (95% confidential interval: 1.71-7.98) compared to those without hypogonadism or with treated hypogonadism. Furthermore, among patients with hypogonadism aged younger than 50 yrs, those treated with sex hormone (5/51: 10%) suffered less frequently from adrenal crisis than those untreated (7/11: 64%, p = 0.0004). In conclusion, the present study has, for the first time, clarified the risk factors of adrenal crisis. Among them, sex hormone deficiency has an especially important implication because it can be treated by hormone replacement therapy with the hope of reducing the risk of adrenal crisis.

Summary Background It has been argued that the best method of acquiring clinical reasoning is through seeing new out-patients. The purpose of this interventional study was to establish a clinical clerkship course for Japanese medical students in an out-patient care setting, with multiple opportunities for reflective practice and improving the clinical reasoning abilities of the students. The effectiveness of the course was also examined. Methods Students performed examinations of new patients and made diagnostic decisions in 20 minutes. They presented their case using the SNAPPS (Summarize the case, Narrow the differential, Analyze the differential, Probe the preceptor, Plan management, and Select an issue for self directed learning) method, and this was followed by feedback from faculty members using the 1–minute preceptor method and a mini clinical evaluation exercise (mini-CEX). Students' clinical reasoning abilities were assessed by the objective structured clinical examination (OSCE) and the script concordance test (SCT). Students' written comments and responses to an interview about the course were also analysed. Cross-sectional data were examined by comparing individual OSCE and SCT scores, and the multiple-choice question examination (MCQ) completed by students who did and did not participate in this project. Results Students in the programme had higher scores on the mini-CEX in all areas. The SCT and OSCE scores were also significantly higher than the scores for the control group. Students' comments about the course, which provided an opportunity for daily reflection, were positive. Discussion Students rapidly acquired clinical reasoning skills through reflective practice. Students also demonstrated motivation to learn through the examination of new patients. The clinical clerkship programme with multiple opportunities for reflective practice in an out-patient care setting substantially improved the clinical reasoning abilities of medical students.

A large number of constituents, such as growth factors, cytokines, and vasoregulatory molecules, contribute a network of cellular interactions to atherosclerotic lesions, and current evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of these constituents. We conducted this study to determine whether GM-CSF has an effect on the fate and function of macrophages. We examined the effect of GM-CSF on macrophages in vitro with a highly inducible HL60 subclone (HL60/DU-1) that we recently established. HL60 cells have been reported to preserve functional GM-CSF receptors, but a GM-CSF allele was rearranged and partially deleted. HL60/DU-1 cells were devoid of GM-CSF immunoreactivity and of autocrine stimulation of GM-CSF. HL60/DU-1 cells fated to die soon after terminal differentiation of macrophages by 1, 25-dihydroxy vitamin D(3) treatment. We found cell death to be mediated mainly by necrosis, not apoptosis, as confirmed by DNA fragmentation in agarose gel electrophoresis, morphological observation under a fluorescence microscope, and assay of lactate dehydrogenase release. Exogeneously administered GM-CSF rescued cells from necrotic death and caused them to survive and generate superoxide anions. We also conducted immunohistochemical analysis on an atherosclerotic human artery. Macrophages, endothelial cells, and smooth muscle cells were found to be GM-CSF positive in an atherosclerotic lesion. In summary, GM-CSF, which is produced by macrophages, endothelial cells, and smooth muscle cells, is thought to act in an autocrine and a paracrine fashion as a necrosis-inhibiting factor against arterial macrophages. This unique function may play an important role in ensuring survival and promoting function in atherosclerotic lesions.

Standard therapy for ACTH-independent bilateral macronodular adrenocortical hyperplasia (AIMAH), a rare form of Cushing's syndrome, is bilateral adrenalectomy. Patients with AIMAH are usually elderly, with a variety of complications, and at risk for surgery. Postoperatively, they must receive lifelong corticosteroids and spend the remainder of their lives avoiding adrenal crisis. Therapy using metyrapone, a potent inhibitor of steroidogenesis, provides the advantages of avoiding the surgery. Its effectiveness is further anticipated because adrenal steroidogenic enzymes are reportedly weak in AIMAH. Treatment with metyrapone thus appears a good therapy for AIMAH, but its effectiveness has not, to our knowledge, been studied. We treated a 59-year-old man with AIMAH with metyrapone. At a low dose of metyrapone (500 to 750 mg/day), his plasma cortisol levels decreased to the normal range, and hypertension and diabetes mellitus were ameliorated. Therapy using metyrapone thus appears effective in treating AIMAH, and can be recommended for high risk AIMAH patients as an alternative therapy.

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.

OBJECTIVE Adrenocortical cells change with age in both subcellular morphology and level of steroidogenesis. This study evaluates the influence of aging on hypercortisolism and clinical manifestations in patients with ACTH-independent Cushing's syndrome due to a cortisol-secreting adrenal adenoma (CS). DESIGN Restrospective study. PATIENTS Thirty-six (33 females and 3 males) with CS. 31 healthy controls were age- and sex-matched to CS patients. MEASUREMENT Patient age at diagnosis was compared to the degree of hypercortisolism and clinical manifestations. The degree of hypercortisolism was estimated using serum cortisol levels and urinary free cortisol excretion. Positive clinical manifestations were quantified using 12 symptoms and signs which resulted in a clinical score for each patient. Endogenous creatinine clearance, estimated disease duration and gender were analysed as possible factors affecting adenoma-secreated cortisol. RESULTS Patient age correlated negatively with serum cortisol level (r = -0.417, P = 0.0107, n = 36) and urinary excretion of free cortisol (r = -0.613, P = 0.0002, n = 31). Analysis of Spearman's rank correlation showed that the clinical score decreased with age (rho = -0.631, P = 0.0004, n = 33). Oedema, weakness/myopathy, hirsutism, striae and psychological changes were infrequent symptoms in the elderly. Using stepwise multiple regression analysis, we estimated the clinical score (Y) by factors of serum cortisol levels (X1:nmol/l), age (X2: years of age) and gender (X3: gender; female = 1, male = 0), i.e. Y = 4.771 + 0.004 X1 - 0.064 X2 + 2.548 X3 (r = 0.743, P < 0.0001, n = 33). Results suggested that aging both suppressed cortisol secretion and repressed manifestations directly as an independent factor, although its direct effect may be weak. CONCLUSIONS Our findings indicated the importance of age as a factor modulating hormonal levels and possible clinical manifestations in patients with cortisol-secreting adenomas.

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.

褐色細胞腫は二次性高血圧をおこす代表的疾患であり,しばしば著しい血圧の変動を呈することで有名である.高血圧は腫瘍からのカテコールアミン過剰分泌が第一の原因である.しかし腫瘍から放出されたカテコールアミンは交感神経終末に取り込まれ,神経インパルスによりそこから放出される機序がある.これが血圧変動の一因となつている.さらに腫瘍からは機械的圧迫,薬物などの誘因によりカテコールアミンが放出され血圧の変動を来たす.またカテコールアミン過剰により受容体数が減少し感受性の低下がおき起立時の低血圧がおきやすい.高血圧クリーゼは致命的なこともあり,速やかな診断と治療が必要である. α1受容体遮断薬であるレギチーンの静脈投与が基本となる.高血圧発作/高血圧クリーゼの病態,診断および治療について紹介する.

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 present two patients with Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia who showed marked plasma cortisol response to lysine-8-vasopressin (LVP) injection (from 930 and 731 pmol/L to 2177 and 1920 pmol/L, respectively), while plasma ACTH levels remained undetectable. The ACTH independence of cortisol secretion in the two patients was determined from the following endocrinological findings. Plasma cortisol levels were not increased by corticotropin-releasing hormone injections and were not suppressed by high dose (16 mg) dexamethasone administrations. The plasma ACTH levels, measured by two independent sensitive immunoassays, were persistently undetectable even after corticotropin-releasing hormone injection, metyrapone administration, and bilateral adrenalectomy. The particular pathological finding of the two cases, atrophic lesions in nonnodular parts of the adrenal cortexes, also indicated ACTH independence of the macronodular hyperplasia. In vitro examination revealed a direct effect of LVP on cortisol secretion from the adrenal cells of the macronodules. We also examined seven patients with Cushing's syndrome caused by adrenal adenoma and found a statistically significant plasma cortisol response to LVP injection. The direct effect of LVP was also demonstrated in cultured adenoma cells. In conclusion, we discovered a direct adrenal effect of LVP on cortisol secretion in patients with ACTH-independent macronodular hyperplasia and, to a lesser extent, in patients with cortisol-producing adrenal adenoma. The cortisol response to LVP may serve to facilitate their diagnosis and choice of therapy.

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)

Patients with Addison's disease, newly diagnosed in the last five years were surveyed nationwide in conjunction with two past surveys. The decreasing incidence, i.e., 168 patients from 1977 to 1981 (survey 1), 108 from 1982 to 1986 (survey 2), and 74 from 1987 to 1991 (the present survey), reflects a decrease in the incidence (n=28) of tuberculous Addison's disease, but not that (n=36) of idiopathic Addison's, making the idiopathic form predominant for the first time in the history of the disease in Japan. Because the interval between the preceding tuberculosis and the onset of Addison's averages 32±15 years, tuberculous Addison's has a relatively late onset. Pigmentation is the most frequent symptom (90%). Other symptoms, abnormal laboratory data, and positive adrenal antibodies are observed less frequently in Japan than in North America or Europe. The types of associated disorders are also distributed differently. Adrenal CT and MRI scans have been the most useful means in differentiating between the two major types of the disease. This report focuses on features distinguishing Japanese sufferers from their western counterparts.(Internal Medicine 33: 602-606, 1994)

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.

Recent findings that LH stimulates renal growth have prompted us to examine whether LH exercises renotropic effects on the growing kidney following uninephrectomy. Intact (not castrated) male rats were used for sham uninephrectomy to induce surgical stress suppressing plasma LH and testosterone levels. Compared to sham surgery, uninephrectomy caused a transient 240% increase in plasma LH with a transient increase in plasma testosterone (80%) and in prostate weight on day 2. Although pituitary LH content did not change, chromatofocusing of pituitary extract revealed changed distribution patterns among seven LH isoforms, suggesting selective synthesis or secretion of LH isoforms. Castrated rats were then used so that surgical stress did not affect LH. Uninephrectomy caused sustained increases in plasma LH levels (60-110% increase) for up to six days. Three days after uninephrectomy, pituitary LH content increased significantly (30% increase) with a change in chromatofocusing profiles of LH isoforms. Plasma and pituitary FSH levels did not change. In summary, uninephrectomy changed LH isoforms quantitatively and qualitatively. These changes imply a change in gonadotropic and renotropic activities of LH after uninephrectomy.

A 47-year-old man had surgery for paraaortic paraganglioma in 1980 and 1985. In 1987, his urinary excretion of catecholamines and metabolites was extremely high. Scintigraphy with 131I-metaiodobenzylguanidine (MIBG) showed multiple bone and liver metastases. He was treated twice with infusions of 3.7 GBq of 131I-MIBG. After the first treatment, he had transient hypertension and pain in the back and right leg. Subsequent 1311I-MIBG scintigraphy showed that the number of metastatic tumors had decreased. The second treatment was less effective. Excess catecholamines were treated with alpha-methyl-p-tyrosine (MPT), a catecholamine synthesis inhibitor, at doses between 250 and 2000 mg/day, which significantly decreased urinary NE excretion. This is the first case treated with 131I-MIBG in Japan.

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.

A 66-year-old woman had massive bilateral adrenal macronodular hyperplasia, found incidentally on an abdominal ultrasonogram. Her plasma ACTH and serum cortisol levels were normal, but they were not suppressed by low-dose dexamethasone. The patient did not exhibit any typical signs or symptoms of Cushing's disease. MRI showed no evidence of a tumor in the pituitary gland. A diagnosis of preclinical Cushing's disease was made, and she was treated with 11-hydroxylase inhibitor metyrapone. As the dose of metyrapone was increased, plasma ACTH levels gradually increased. After three years of treatment, she developed moon-face. Her plasma ACTH and serum cortisol concentrations were at their highest levels. A pituitary microadenoma was detected by MRI, whose source of ACTH was demonstrated by the definite step-up of central/peripheral ratio of ACTH obtained by cavernous sinus sampling. Overt Cushing's disease was diagnosed, and a pituitary tumor was removed by transsphenoidal surgery. In conclusion, the clinically and endocrinologically overt Cushing's disease characterized by macronodular adrenal hyperplasia was converted from a preclinical form. This case offers some insight into the clinical and biological features of preclinical Cushing's disease.

Ampulla cardiomyopathy is named after the echocardiographic abnormalities occurring in this condition, characterized by extensive akinesis (ballooning ) of the apical region with hypercontraction of the basal segment of the ventricle. We describe 3 young female anorexia nervosa patients showing evidence of this cardiac complication after hypoglycemia. One case was complicated by echocardiographically confirmed ampulla cardiomyopathy while the other 2 patients showed increases in myocardial enzymes and transient electrocardiographic abnormalities consistent with this complication. The precipitating event for all three patients was hypoglycemic coma, and this is the first case report in which this factor lead to the complication of ampulla cardiomyopathy in anorexia nervosa patients.

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.