Production of parathyroid hormone and parathyroid-hormone-related protein by breast cancer cells in culture

Parathyroid-hormone-related protein (PTHrP) has been implicated in the origin of malignant hypercalcaemia. However, PTHrP production is not restricted to neoplastic cells, it is widespread among a variety of normal cell types and tissues. A physiological role for PTHrP has not been well defined. We describe a case of breast cancer with bone metastases and humoral hypercalcaemia of malignancy, with high levels of plasma C-terminal parathyroid hormone (PTH), mid-molecule PTH and PTHrP. Cells from breast cancer biopsies were cultured and medium samples assayed for the C-terminal and mid-molecule fragments, intact PTH and PTHrP. The data indicate a progressive increase in both PTH fragments and PTHrP levels, over a period of 30 days. No temporal parallelism exists between PTH fragments and PTHrP concentrations, the former being maximum at the 14th day, and the latter at the 30th day from the beginning of the culture. Our results indicate a coproduction of PTH and PTHrP by the breast cancer cells both in vivo and in vitro.Abbreviations PTH parathyroid hormone - PTHrP PTH-related protein - HHM humoral hypercalcaemia of malignancy This work was supported by a grant awarded by the Italian National Research Council (CNR): Clinical Applications of Oncology Research (ACRO) no. 92.02170.PF/39     

Calcium homeostasis: reassessment of the actions of parathyroid hormone

The purpose of this report is to adjust our interpretation of the actions of parathyroid hormone (PTH) to include its action on the bone mineral-noncollagenous protein interactions at all bone surfaces. The three primary areas that respond to PTH are: (1) all bone surface areas in contact with the extracellular fluid (ECF), (2) the kidney, and indirectly the intestinal tract, and (3) the bone remodeling sequence. The primary rapid action of the hormone is to set and maintain the free calcium concentration of the ECF. This it does by raising the equilibrium level at bone mineral surfaces. It affects the noncollagenous protein bone mineral process to raise the free calcium level in the ECF from the base level of 3.5mg/100ml to the physiological level of 5.0mg/100ml. Maintaining the higher level requires continuous secretion of parathyroid hormone. The action of PTH at bone surfaces tends to be catabolic in nature in regard to bone loss. The hormone also acts on the kidney to raise the threshold for calcium reabsorption and by stimulation of renal hydroxylation of vitamin D to increase intestinal absorption of calcium. Its action here is to supply the ECF with calcium derived from food intake. This is the extent of PTH action on renal processes. PTH acts on all three steps in the bone remodeling process. While its total function here is not clear, the result is a net increase in the synthesis of collagen. The report concludes by comparing the actions of PTH as proposed here to the functions of PTH that have been proposed in the past.     

原发性胆汁性肝硬化伴甲状旁腺腺瘤诊治

原发性胆汁性肝硬化可从多种途径影响钙和维生素D的代谢,进而引起骨软化和继发性甲状旁腺功能亢进症,但当同时出现低蛋白血症时可掩盖血钙的真实水平.因此若该病合并甲状旁腺腺瘤时容易漏诊.本文详细介绍1例原发性胆汁性肝硬化合并甲状旁腺腺瘤的诊治过程,旨在提醒临床医生重视低蛋白血症对血清总钙水平的影响.     

甲状旁腺素受体1表达对高糖状态下INS-1细胞功能影响研究

目的 观察甲状旁腺受体1(PTH1R)的表达对胰岛β细胞胰岛素合成与分泌功能影响.方法 构建出PTH1R基因沉默的细胞模型后,分别应用放射免疫法观察25 mmol/L D-葡萄糖处理后对照组、阴性基因克隆组(siPTH1R-NC)、PTHrP组及阳性基因序列组(siPTH1R)细胞内胰岛素含量、葡萄糖刺激胰岛素分泌能力、应用Fluo-3/AM 检测INS-1细胞内钙离子浓度及应用INS-1细胞2-脱氧-[3H]-葡萄糖摄入率检测INS-1细胞葡萄糖转运能力.结果 PTHrP组胰岛素分泌能力高于其他3组,siPTH1R组则低于对照组及siPTH1R-NC组(均P＜0.01);PTHrP组中细胞内胰岛素含量显著高于对照组、siPTH1R-NC及siPTH1R组(均P＜0.01),其他3组间差异无统计学意义;PTHrP组中钙离子浓度水平高于其他3组,siPTH1R组低于对照组及siPTH1R-NC组.PTHrP组中细胞2-脱氧-[3H]-葡萄糖摄入率高于其他3组.结论 高糖状态下PTH1R表达水平与INS-1细胞胰岛素合成与分泌功能有关,可能为INS-1细胞自我保护的一种作用.

Abstract：

Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.     

Involvement of parathyroid hormone-related peptide in the aggressive phenotype of colorectal cancer cells

Colorectal cancer (CRC) remains one of the leading causes of mortality from malignant diseases worldwide. In general terms, CRC presents high heterogeneity due to the influence of different genetic and environmental factors; also, the neoplastic cells are strongly influenced by the extracellular matrix and several surrounding cells, known together as the tumor microenvironment (TME). Bidirectional communication takes place between the tumor and the TME through the release of autocrine and paracrine factors. Parathyroid hormone-related peptide (PTHrP) is a cytokine secreted by a wide variety of tissues and is able to regulate several cellular functions both in physiological as well as in pathological processes. It exerts its effects as a paracrine/autocrine factor, although its mode of action is mainly paracrine. It has been shown that this peptide is expressed by several tumors and that the tumor secretion of PTHrP is responsible for the malignant humoral hypercalcemia. Eight years ago, when our research group started studying PTHrP effects in the experimental models derived from intestinal tumors, the literature available at the time addressing the effects of PTHrP on colorectal tumors was limited, and no articles had been published regarding to the paracrine action of PTHrP in CRC cells. Based on this and on our previous findings regarding the role of PTH in CRC cells, our purpose in recent years has been to explore the role of PTHrP in CRC. We analyzed the behavior of CRC cells treated with exogenous PTHrP, focalizing in the study of the following events: Survival, cell cycle progression and proliferation, migration, chemoresistance, tumor-associated angiogenesis, epithelial to mesenchymal transition program and other events also associated with invasion, such us the induction of cancer stem cells features. This work summarizes the major findings obtained by our investigation group using in vitro and in vivo CRC models that evidence the participation of PTHrP in the acquisition of an aggressive phenotype of CRC cells and the molecular mechanisms involved in these processes. Recently, we found that this cytokine induces this malignant behavior not only by its direct action on these intestinal cells but also through its influence on cells derived from TME, promoting a communication between CRC cells and surrounding cells that contributes to the molecular and morphological changes observed in CRC cells. These investigations establish the basis for our next studies in order to address the clinical applicability of our findings. Recognizing the factors and mechanisms that promote invasion in CRC cells, evasion to the cytotoxic effects of current CRC therapies and thus metastasis is decisive for the identification of new markers with the potential to improve early diagnosis and/or to predict prognosis, to predetermine drug resistance and to provide treatment guidelines that include targeted therapies for this disease.     

Effect of chronic ethanol consumption on the response of parathyroid hormone to hypocalcemia in the pregnant rat

BACKGROUND: Chronic alcohol (ethanol) consumption during pregnancy results in maternal/fetal hypocalcemia, which may underlie some of ethanol's adverse effects on maternal and fetal bone, and fetal/neonatal health. Ethanol appears to alter the relationship between parathyroid hormone (PTH) and blood calcium (Ca) level, and PTH does not increase in response to ethanol-induced hypocalcemia. However, it is not known whether ethanol actually prevents PTH from responding, or whether the ability to regulate blood Ca is intact, but ethanol lowers the level of Ca maintained. The objective of this study was to determine whether chronic ethanol consumption impairs the ability of the pregnant female to increase PTH in response to acute hypocalcemia. METHODS: Rats were fed isocaloric diets with ethanol (36% ethanol-derived calories, E group) or without ethanol [pair-fed (PF) and control (C) groups], before and throughout 21 days of gestation. On day 21 gestation, rats received an intraperitoneal injection of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (300 or 500 mumol/kg body weight) or saline (saline group), or no injection (baseline group). Blood was collected from the baseline group, and at 30 or 60 minutes postinjection (saline and EGTA groups), and analyzed for ionized Ca (iCa), pH, and PTH. RESULTS: Consistent with previous studies, ethanol consumption decreased blood iCa levels at baseline, but PTH levels did not differ among groups. Administration of EGTA significantly decreased blood iCa levels by 30 minutes, but ethanol did not prevent PTH from increasing in response to the hypocalcemia. In all diet groups, PTH levels were significantly increased by 30 minutes. Ethanol did, however, appear to decrease the maximum PTH level achievable in blood. CONCLUSIONS: These data suggest that chronic ethanol consumption does not impair the ability of the pregnant rat to raise serum PTH levels in response to acute hypocalcemia, but ethanol's effect on maximal PTH secretion could impair the ability of the pregnant female to sustain high PTH levels in response to chronic hypocalcemia.     

The concentration of free calcium in plasma is set by the extracellular action of noncollagenous proteins and hydroxyapatite

The late Wm. F. Neuman frequently included the following statement in his speeches: “Plasma calcium is undersaturated in respect to itself, but supersaturated in respect to bone”. As a physical chemist he knew that if plasma or extracellular fluid came in direct contact with bone surfaces plasma calcium level should fall to the saturated solubility of hydroxyapatite. How could the condition given in the quote exist? He answered this question by laboratory experiments but unfortunately died before he could explain it scientifically. In the current perspective article we feel that we cannot only explain Neuman’s riddle but also use the answer to describe a revised premise for extracellular control of calcium concentrations in body fluids. The answer lies in the solubility of hydroxyapatite. A simple contact of bone mineral surface with body fluids should lead to a calcium concentration in plasma too low to support life. The evolutionary process prevented this by adding one or more noncollagenous proteins at the surface of hydroxyapatite. These proteins elevated the saturated solubility of the crystal latticework sufficiently to provide a calcium concentration that would sustain life. This report explains the solubility process in as much detail as our scientific information will permit and also includes the role of parathyroid hormone in the process. We hope that serious study of our article will permit acceptance of our premise for calcium control and induce further study that should demonstrate its importance in all aspects of bone metabolism.     

Sequential determination of calcium distribution in B cells at the various phases of glucose-induced insulin secretion

Summary Localization and quantification of calcium pyroantimonate precipitates within the B cells, and determination of insulin secretion were performed in rat pancreas perfused with 3.3 and 16.6 mmol/l glucose. Observations were carried out during the peak, the refractory period, and at 10 and 20 min in the second phase of glucose secretion after the start of a glucose challenge. Specific calcium pyroantimonate precipitates, assessed by EGTA cross-incubation, appeared attached to plasma membrane, Golgi complex, mitochondria, cytoplasmic matrix and secretory granules. The total number of cellular calcium pyroantimonate precipitates increased with perfusion time, being significantly higher at every time-point with the higher concentration of glucose (16.6 mmol/l) than with the 3.3 mmol/l glucose concentration. Calcium pyroantimonate precipitates showed a progressive increment both in plasma membranes and mitochondria. In the cytoplasmic matrix, B granules and Golgi complex, a sharp increase in the number of precipitates was detected at the refractory period, followed by a continuous decrease until the end of the experiment. These results show that the number of calcium pyroantimonate precipitates, localized in different organelles, changes according to the functional state of B cells. They stress the importance of intracellular readily exchangeable pools as regulators of calcium availability for insulin stimulus-secretion coupling.     

The set point for maternal glucose homeostasis is lowered during late pregnancy in the rat: the role of the islet beta-cell and liver

Summary The aim of this study was to determine the effects of late pregnancy on the ability of insulin to suppress maternal hepatic glucose production in the rat. Unlike in most previous studies, suppression of hepatic glucose production was measured at levels of glycaemia above the relatively hypoglycaemic basal pregnant level. Glucose kinetics were measured using steady-state tracer methodology in chronically catheterised, conscious virgin control and pregnant rats, firstly, during basal and low-dose hyperinsulinaemic euglycaemic clamp conditions and secondly, during a three-step glucose infusion protocol (glucose infusion rates of 0, 60 and 150 μmol · kg⁻¹· min⁻¹). During the clamps, plasma glucose levels were not different (6.1 ± 0.4 vs 6.5 ± 0.3 mmol/l, pregnant vs virgin; N. S.), but plasma insulin levels were higher in the pregnant rats (242 ± 30 vs 154 ± 18 pmol/l, pregnant vs virgin; p < 0.05) most probably due to stimulated endogenous insulin release in this group. Hepatic glucose production was suppressed from basal levels by 41 % in virgin and 90 % in pregnant rats. During the glucose infusion studies, at matched insulin levels (147 ± 10 vs 152 ± 14 pmol/l), but at plasma glucose levels which were much lower in the pregnant rats (5.5 ± 0.2 vs 8.4 ± 0.6 mmol/l, pregnant vs virgin; p < 0.0001), hepatic glucose production was shown to be suppressed by a similar degree in both groups (41 ± 5 vs 51 ± 5 % from basal, pregnant vs virgin; N. S.). Both the plasma insulin and percentage suppression of hepatic glucose production dose responses to plasma glucose were markedly shifted to the left indicating that the plasma glucose set point is lowered in pregnancy. In conclusion, suppression of hepatic glucose production by insulin is not impaired and the set point for plasma glucose homeostasis is lowered during late pregnancy in the rat. [Diabetologia (1996) 39: 785–792] Received: 2 October 1995 and in final revised form: 1 February 1996     

Changes in serum phosphate during beta-blockade in healthy men are not due to changes in the renal handling of phosphate

Serum parathyroid hormone (PTH), phosphate, ionized calcium, magnesium, alkaline phosphatase and creatinine and the urinary excretion of phosphate and creatinine were studied after 1 and 3 weeks beta-blockade in 32 young healthy men given either atenolol at 50 mg, metoprolol at 100 mg, propranolol at 80 mg or placebo twice a day. After 1 week treatment serum phosphate (mean (range] increased in the propranolol-treated subjects (1.17 (0.99-1.30) to 1.32 (1.08-1.71) mmol l-1 (P = 0.03), minor changes were found in the atenolol and the metoprolol-treated subjects but not in the placebo group. Serum phosphate was unchanged compared to pretreatment values after 3 weeks. Renal clearance and urinary excretion of phosphate and creatinine was unchanged after both 1 and 3-weeks treatment. Serum PTH, ionized calcium, magnesium and alkaline phosphatase were unchanged in all groups, whereas serum urate and creatinine increased in the metoprolol-treated subjects after both 1 and 3 weeks.     

Binding of [3H]ouabain to endothelial cells derived from various vascular beds

Summary Binding experiments were performed with [³H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [³H]ouabain binding to endothelial cells form pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (K_D) of 3.29±0.31 nmol/l and a binding capacity (B_max) of 5.22±0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [³H]ouabain revealed much lower affinity (K_D 95±15 nmol/l, B_max 2.08±0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na⁺/K⁺-ATPase (proscillaridin A>ouabain>digoxin>g-strophanthidin>digoxigenin>dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K⁺ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na⁺/K⁺-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg²⁺-ATPase activity.     

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin–radixin–moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1–36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1–36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1–36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27^kip1 and the anti-proliferative effect of PTHrP(1–36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.     

Complement action on secretory cells identified by the reverse hemolytic plaque assay: modified assay eliminates exposure of secretory cells to complement

The application of a hemolytic plaque assay to antigen-secreting endocrine cells has brought about great advances in the study of regulation of hormone secretion. The reverse hemolytic plaque assay (RHPA) has enabled quantitation of secretion at the single cell level with simultaneous analysis of the population response. Moreover it has allowed unambiguous identification of specific cell types in mixed cell populations while maintaining the viability of the cells for further physiological experiments. Concern has arisen, however, regarding potential complement attack on those cells of interest, causing sublytic permeabilization leading to altered physiological function. To test this possibility, prolactin release from dispersed anterior pituitary cells was quantitated in two protocols of the RHPA. Cells were exposed to complement either subsequent to the termination of antiserum incubation or simultaneously with antiserum incubation, during which time hormone release is being detected. The presence of complement during antiserum incubation resulted in significant increases in mean plaque area as compared to the separate incubation procedure (13 709 ± 698vs 9251 ± 547 μm²). Analysis of the population profile of plaques indicated that the increased mean plaque area reflected a rightward shift in the frequency distribution of plaque size. The general increase in hormone release in the antibody/complement group is consistent with a predicted permeabilizing action of the complement on the secretory cells. To avoid this potentially damaging effect of complement on secretory cells to be used in subsequent physiological experiments, we have developed a modification of the RHPA in which the secretory cells are unequivocally identified without being exposed to complement.     

Studies of the biological and immunological properties of parathyroid hormone, labeled selectively on the methionine residues by [3H]methyl exchange to high specific activity.

A technique is described for labeling bovine parathyroid hormone (bPTH) with tritium by [3Hmethyl exchange. The methionine residues were first methylated with [3H]methyl iodide at pH 4, and the reaction products were separated by cation exchange chromatography. The major peak consisted to hormone in which both methionines were converted to [3H]methyl methionine sulfonium iodide (3H-methylated bPTH). This product was then demethylated with 2-mercaptoethanol (6 M) at pH 8.6 to regenerate the hormone in an unmodified but tritiated form ([3H]bPTH), with a specific activity of 1.7 Ci/mmol. High pressure liquid chromatographic analysis showed that 96% of the radioactivity was incorporated into the methionine residues. There was no evidence of any alteration in the primary structure, as [3H]bPTH was found to run in the same position as unlabeled bPTH on cation exchange chromatography and disc gel electrophoresis and to have an identical absorption spectrum in the 240- to 330-nm range. Moreover, [3H]bPTH had full biological activity, as measured by an in vitro bioassay based on activation of rat renal cortical adenylate cyclase, although 3H-methylated bPTH was almost completely inactive. Similarly, while 3H-methylated bPTH had reduced potency in a RIA specific for antigenic sites in the 1--34 region of the sequence, [3H]bPTH was found to have full activity. The preparation of labeled bPTH was repeated using [14C]methyl iodide, with similar results, although [14C]bPTH was found to have somewhat reduced immunological and biological activities. While [3H]bPTH had a lower specific activity than can be obtained by various other techniques for incorporating tritium or 125I into peptides, biosynthetic labeling is at present the only alternative method for preparing biologically active, labeled bPTH without altering the primary structure. By comparison with this technique, the present method gave a product of a much higher specific activity which was labeled specifically in the biologically essential amino-terminal region. The same simple chemical procedures are clearly of wide potential application to the preparation of other labeled peptides.     

Selected properties of [3H]prostaglandin E1 binding to dispersed bovine luteal cells.

Selected properties of [3H]prostaglandin (PG) E1 binding to collagenase dispersed bovine luteal cells were studied and compared with those observed in luteal plasma membranes. [3H]-PGE1 specific binding to a relatively homogeneous population of luteal cells was a rapid (K1 = 4.2 X 10(5) M-1 .sec-1), reversible (K-1 = 3.9 X 10(-3) sec-1), saturable and specific process at 38 degrees C. The binding was homogeneous with an apparent dissociation constant of 2.4 nM and 1.8 X 10(5) receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [3H]PGE1 binding in a dose-dependent manner. The potency order for this inhibition of binding was: PGE 2 greater than PGE1, (15S)-15-methyl-PGE2 methyl ester greater than PGF2alpha greater than PGF1alpha greater than other PGs, PGE, PGF metabolites and PGF analogs. Other than the homogeneous nature of [3H]PGE1 binding and the greater effectiveness of PGE2 compared to PGE1 in cells, the rest of the properties of [3H]PGE1 binding to cells were in excellent agreement with those observed in plasma membranes.     

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

The Legionella pneumophila Dot/Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot/Icm system. In this study, the mechanism of Dot/Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot/Icm system. A leucine (L372) residue at the -3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot/Icm system, most other Dot/Icm substrates were found to have amino acid residues with similar physical properties at their -3 or -4 C-terminal positions. These data demonstrate that the Dot/Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.     

In vivo incorporation of [3H]leucine and [3H]tyrosine by caudal neurosecretory cells of the trout Salvelinus fontinalis in relation to osmotic manipulations. A radioautographic study

The in vivo incorporation of tritiated leucine and tyrosine during various time intervals by caudal neurosecretory cells of trout Salvelinus fontinalis was studied quantitatively with light microscopic radioautography in relation to osmotic manipulations. The [³H]leucine labeling in fish exposed to deionized water for 3 days was much higher than in fish kept in fresh water, indicating enhanced synthetic activity. Under both conditions, the maximum of incorporation was observed 12 hr after injection of the tracer. The reduced radioautographic reaction noted at 24 hr suggested a rapid transit of newly synthesized proteinaceous material from cell body cytoplasm toward the axonal compartment. [³H]Tyrosine was incorporated to a greater degree than [³H]leucine in cells of fish in fresh water. The varied synthetic activities exhibited by caudal neurosecretory cells in response to osmotic stimuli indicate possible participation by these cells in osmotic and ionic regulation.     

Murine natural killer T(NKT) cells [correction of natural killer cells] contribute to the granulomatous reaction caused by mycobacterial cell walls

Mice injected with deproteinized cell walls prepared from the strain H37rv of Mycobacterium tuberculosis develop a granuloma-like lesion in which NKT cells are predominant. NKT cells play a primary role in the granulomatous response, because the latter does not occur in Jalpha281(-/-) mice, which miss NKT cells. The glycolipidic fraction of the cell walls is responsible for the recruitment of NKT cells; the recruiting activity is associated with fractions containing phosphatidylinositolmannosides. These results define a powerful experimental set up for studying the in vivo induction of NKT cell responses to microbial components.     

Severe combined immunodeficiency, interleukin-2 (IL-2), and the IL-2 receptor: experiments of nature continue to point the way [see comments]

The recent discovery of molecular defects in three forms of X-linked immunodeficiency has quickly transformed the study of immunodeficiency into one of the most exciting in basic and clinical immunology. The identification of defects in the IL-2R gamma chain in the etiology of X- linked SCID has suggested a heretofore unanticipated functional role of the gamma chain in immunologic development. While new and novel cytokines and cytokine receptors continue to be identified, it has become clear that our knowledge of IL-2, one of the best understood cytokine/receptor systems, is far from complete. Clarifying the molecular interactions between IL-2 and its receptor complex will improve the sophistication with which these interactions are manipulated in the clinic for the treatment of autoimmune disorders and allograft rejection, treatment of lymphoid malignancies, and cytokine- based therapies for immunotherapeutic treatment of nonlymphoid cancers. Recent gene therapy approaches to the treatment of children with the ADA-deficient form of SCID offers yet another exciting path for investigation. The use of retrovirally infected cord blood hematopoietic progenitor cells in attempts to reconstitute the immune system of ADA-deficient SCID children with ADA-producing cells raises the possibility of similarly "correcting" the defect in X-linked SCID. Such approaches almost certainly loom on the near horizon for other diseases. However, in view of the complexity and potentially pleiomorphic nature of defects in the IL-2R gamma chain, both in terms of their identification and correction, gene therapy for treatment of X- linked SCID will require a thorough understanding of the molecular nature of the respective defects. Effective therapy will require precise knowledge of the defects, in terms of their influence on the ligand, receptor, and signaling apparatus, as well as their potential effects on cells of multiple lineages. However, these caveats aside, the potential for understanding and correcting a disease that robs infants at so early an age of the potential for a normal life will continue to make these exciting and extraordinarily rewarding pursuits.