Impact of the structures of macrocyclic Michael acceptors on covalent proteasome inhibition.

Molecules that have a reactive functional group within a macrocycle represent a class of covalent inhibitor. The relationship between reactivity and affinity for the target is cooperative and complicated. An understanding and characterization of this class of inhibitor are vital for the development of covalent inhibitors as drug candidates. Herein, we describe a systematic analysis of structure-activity relationships using a series of syringolin analogues, which are irreversible covalent inhibitors of proteasomes. We investigate the detailed mechanistic effects of the macrocycles on affinity and reaction rate.

Musculoskeletal symptoms and neurological investigations in adrenocortical insufficiency: a case report and literature review.

OBJECTIVES: Various forms of adrenocortical insufficiency can cause musculoskeletal symptoms such as muscle pain, tautness of the limbs, arthralgia, and flexion contractures. However, the findings of neurological investigations are inconclusive and have not been well summarized. METHODS: We report the case of a 61-year-old man with isolated adrenocorticotropic hormone deficiency who presented with musculoskeletal symptoms, including flexion contractures. We performed three neurological investigations: nerve conduction studies, electromyography, and muscle biopsy analysis. Further, we reviewed reports of 16 patients with various forms of adrenocortical insufficiency and musculoskeletal symptoms by considering the findings of these three investigations. RESULTS: From the literature review, we found that (a) analysis of muscle biopsy is the most sensitive technique, followed by electromyography and then nerve conduction studies; and (b) the longer the duration of the musculoskeletal symptoms, the greater the incidence of abnormal findings with all three techniques. CONCLUSIONS: Physicians may prioritize neurological investigations, depending on these findings.

Induction of mononuclear cell infiltration into liver by Japanese herbal medicine.

Juzen-Taiho-To (JTT) is a Japanese herbal medicine that has been administered mainly to patients weakened by long illness. Currently, it has also been used for cancer patients and showed antitumor effects that have been reported as phagocytosis enhancement, cytokine induction and antibody production. In this study, we examined the effect of oral administration of JTT in mice on the immunological restoration of the liver, especially focused on natural killer (NK) T-cell induction. Mice were grouped to receive JTT or placebo orally for a period of 1, 3 and 7 days. After sacrifice, the liver tissue was fixed, embedded and stained with hematoxylineosin and some antibodies by common staining methods. Transmission electron microscope (TEM) observation was also carried out. Although the JTT-treated mice had the same appearance as the non-JTT-treated mice, their livers were infiltrated by massive mononuclear cells, some of which were aggregated in clusters. Immunohistochemical staining revealed that there was abundant cytokine expression of interleukin (IL)-12 and massive infiltration of mononuclear cells with large granules in the liver of JTT-treated mice. Oral administration of JTT may induce the expression of IL-12 and be followed by immunological restoration such as NK T-cell induction in liver

Human ACTH hypersensitivity syndrome associated with abnormalities of the ACTH receptor gene.

OBJECTIVE: Activating mutations of the ACTH receptor have not been previously described. We investigated a 69-year-old woman with normal blood cortisol but undetectable blood ACTH concentrations. The aim of this study was to evaluate her hypothalamo-pituitary-adrenal axis by measuring circadian variation in blood ACTH and cortisol, and by performing CRH and ACTH stimulation and dexamethasone suppression tests. We also examined biological activity of her circulating blood ACTH using bovine adrenocortical cell suspensions and ACTH receptor gene structure by Northern blotting analysis. RESULTS: Random plasma cortisol concentrations ranged from 182 to 328 nmol/l, while ACTH concentrations were always undetectable. After an intravenous bolus injection of human CRH 100 micrograms, plasma ACTH rose slightly, while plasma cortisol increased appropriately. ACTH stimulation tests revealed that a small amount of ACTH (5 ng/kg b.w.) had the maximal cortisol stimulatory activity, and even smaller amounts of ACTH (0.5 and 0.05 ng/kg b.w.) produced significant increases in cortisol levels. ACTH bioassay of the patient's plasma demonstrated weak biological activity in the HPLC fractions which corresponded to the band of synthetic human ACTH 1-39. The ACTH receptor coding region was amplified by polymerase chain reaction using the leucocyte genomic DNA. There were two base mutations; cysteine 21-->arginine and serine 247-->glycine in the sequences coding for the first extramembranous N-terminal domain and the third extramembranous loop of the ACTH receptor. CONCLUSIONS: This patient with normal blood cortisol but undetectable ACTH levels showed increased adrenocortical sensitivity to ACTH and two point mutations in the ACTH receptor gene. This study, therefore, reports a previously undescribed syndrome--ACTH hypersensitivity syndrome--and provides insights into the molecular mechanism of ACTH receptor action.

Differences in down-regulation of glucocorticoid receptor mRNA by cortisol, prednisolone and dexamethasone in HeLa cells.

Glucocorticoids regulate the levels of their cognate receptors in a number of target tissues and in many different cell lines. We have compared the effect of three glucocorticoids, cortisol and its synthetic derivatives, prednisolone an dexamethasone, on the levels of glucocorticoid receptor (GR) mRNA in HeLa cells. Clinically, the synthetic derivatives are more active in hormonal action and have a longer half-life than cortisol. In the present study, the amounts of GR mRNA in HeLa cells were examined by Northern blot hybridization after treatment with cortisol, prednisolone or dexamethasone. These glucocorticoids decreased GR mRNA levels differently. After 24 h treatment with 1 x 10(-5) M cortisol, GR mRNA levels were only marginally suppressed (90% of the control), while prednisolone and dexamethasone suppressed GR mRNA levels to 67 and 57%, respectively. These differences may relate to the biological activities of these glucocorticoids. In time course studies, GR mRNA levels of the cells treated with cortisol and prednisolone decreased to the minimum levels within 4 h and then recovered gradually, while those treated with dexamethasone reached the minimum level at 8 h and remained suppressed for more than 24 h. These differences may relate to the biological half-lives of these glucocorticoids.

Role of the time factor in signaling specificity: application to mitogenic and metabolic signaling by the insulin and insulin-like growth factor-I receptor tyrosine kinases.

The signal transduction pathways activated by hormones, growth factors, and cytokines show an extraordinary degree of cross-talk and redundancy. This review addresses the question of how the specificity conferred at the binding step is maintained through the signaling network despite the convergence of multiple signals on common efferent pathways such as mitogen-activated protein (MAP) kinase. The mechanism of receptor activation by ligand-induced dimerization provides a signaling device with both a switch and a timer. The role of the time factor, ie, of signaling kinetics, as a determinant of selectivity is discussed with emphasis on the receptor tyrosine kinases and cytokine receptors, and especially mitogenic versus metabolic signaling by insulin and insulin-like growth factor-I (IGF-I).

Exacerbation of rheumatoid arthritis after removal of adrenal adenoma in Cushing's syndrome.

A 46-year-old woman with rheumatoid arthritis had been on non-steroidal antiinflammatory agents for eighteen years until she developed cushingoid features and hypertension resistant to antihypertensive drugs. She had high plasma cortisol and 24 h urinary 17-hydroxycorticosteroids (17HCS) which were not suppressed by 8 mg dexamethasone per day for two days. The circadian rhythm of plasma cortisol was absent and plasma ACTH concentrations were suppressed before and after intravenous administration of CRH. Abdominal computed tomography demonstrated a tumor (3.0 x 3.0 x 2.3 cm) in the right adrenal gland and a 131I-6 beta-19-nor-methylcholesterol scan revealed marked uptake on the same side. The patient underwent a right adrenalectomy and the diagnosis of a cortisol secreting benign adenoma was histologically confirmed. Blood pressure declined and cushingoid features regressed, but three months after the operation and while the patient was on replacement, she complained of pain on motion, marked tenderness and swelling of fingers, wrists, elbows, knees and foot joints, and had very high rheumatoid factors. Treatment with immunosuppressive drugs and oral and intraarticular administration of glucocorticoids were necessary to relieve the clinical symptoms of rheumatoid arthritis. In summary, we report a patient with rheumatoid arthritis and Cushing's syndrome due to an adrenal adenoma, in whom rheumatoid arthritis was exacerbated after curing the Cushing's syndrome. This suggests that it is imperative to follow the development and/or course of autoimmune diseases after the treatment of Cushing's syndrome.

The insulin-like growth factor-I receptor. Structure, ligand-binding mechanism and signal transduction.

The nonclassical binding kinetics of IGF-I and insulin to their respective receptors, suggestive of negative cooperativity, can be readily explained by our recently proposed novel binding mechanism whereby the bivalent ligand bridges the two receptor alpha-subunits alternatively at opposite sites in a symmetrical receptor structure. The bivalent binding mechanism also explains bell-shaped bioactivity curves. The possible role of different binding modes versus differences in downstream signaling by insulin and IGF-I in producing specific mitogenic or metabolic responses is discussed.

[Synthesis and regulation of growth hormone secretion].

Human growth hormone (hGH) is a single chain, 22 kd-protein with two intramolecular disulfide bonds. The hGH gene is located on chromosome 17 at band q22-q24 and has four introns separating five coding exons. The expression of hGH is restricted to the pituitary and regulated by GHF-1 which binds to the hGH promoter acting in concert with several other more ubiquitous DNA binding proteins. The secretion of hGH is regulated by GH releasing hormone (GRH) and somatostatin. GRH controls GH synthesis by stimulating transcription of GH mRNA while somatostatin determines the timing and amplitude of GH pulses. Pulsatile GH secretion is influenced by a number of neurogenic, metabolic and hormonal factors.