37Salmonella strains have recently been developed as antitumor agents capable of both preferentially amplifying within tumors and expressing prodrug-converting enzymes such as the herpes simplex thymidine kinase 1 . These bacteria were attenuated by auxotrophic mutations that limited their pathogenesis in normal tissues but retained high-level replication within the tumors following systemic administration. The auxotrophic requirements of these Salmonella are apparently met within the tumor environment where they then replicate, reaching up to more than 1000 times the concentration found in normal tissues.A significant limitation for safe use of systemically administered bacteria in humans is the ability of the bacteria to induce tumor necrosis factor α (TNFα)-mediated septic shock 2,3 . However, modifications in bacterial components responsible for eliciting host immune responses such as TNFα induction could interfere with tumor targeting or antitumor activity.Several mutations in lipid biosynthesis are known in Escherichia coli and Salmonella sp. that lower TNFα induction and render the bacteria nontoxic. Some mutations, such as kdo -result in the production of lipid IV A , which substantially lowers TNFα induction and acts as an antagonist to the TNFα response from wild-type lipid A 4,5 . However, these and most other lipid mutations are temperature-sensitive and conditionally lethal to the bacteria 6 , limiting the potential for tumor-based amplification seen in auxotrophic Salmonella 1 .In E. coli, the msbB (mlt) gene 7,8 is involved in the terminal myristoylation of lipid A 9,10 . Genetic disruption of this gene in E. coli results in a stable nonconditional mutation that lowers TNFα induction up to 10-fold by whole bacteria or up to 10,000-fold by purified lipopolysaccharide (LPS) 9 . A similar toxicity profile is observed when the msbB gene is disrupted in Salmonella 11 . We generated a deletion in the coding sequence of msbB within a hyperinvasive strain of Salmonella we previously used for tumor-targeting as well as the parental wild type, and examined the effect on virulence and TNFα production both in vitro and in vivo. Results indicate that msbB -mutant Salmonella retain the properties of tumor accumulation and tumor suppression in the absence of eliciting high levels of TNFα. Results Isolation and genetic disruption of the Salmonella msbB gene.DNA sequence analysis of Salmonella msbB clones obtained by DNA/DNA hybridization indicated the presence of an msbB homolog with flanking gene organization (orfU, msbB, pykA, and zwf) identical to E. coli 8 . The DNA homology of the Salmonella msbB and the E. coli msbB was determined to be 75%, and the amino acid homology 98%, confirming that the cloned Salmonella gene is an msbB homolog.Putative knockouts obtained by transformation of the linearized deletion construct were confirmed by several criteria using Southern blot analysis (Fig. 1): Two bands corresponding to the tetracycline gene were observed in the knockout construct and in the knockout clones and w...

The primary structure of ovine hypothalamic hypophysiotropic luteinizing hormone-releasing factor, LRF, has been established as pGlu-His-Trp-SerTyr-Gly-Leu-Arg-Pro-Gly-NH2 by hydrolysis of the peptide with chymotrypsin or pyrrolidone-earboxylylpeptidase and by analysis of the products by an Edman-dansylation sequencing technique, as well as by mass spectrometry of the derived phenylthiohydantoins. A decapeptide with the proposed primary structure, prepared by total synthesis, gave the same result on sequencing. The synthetic decapeptide possesses the same biological activities as the native ovine LRF. The amino-acid sequence of ovine LRF is identical to that already published for porcine LRF.Various areas of the central nervous system participate in the fine regulation of the secretion of all adenohypophysial hormones. The ultimate integrator of information originating in the central nervous system is the hypothalamus. The final information from the hypothalamus to the adenohypophysis is not transmitted in the form of nerve impulses, but is carried in the form of specific hypothalamic hypophysiotropic substances, the hypothalamic releasing factors, that are carried through the hypothalamo-hypophysial portal system of capillaries from the median eminence region of the ventral hypothalamus to the cells of the adenohypophysis. There is good physiological evidence that such a hypothalamic control is involved in the secretion of the gonadotropin, luteinizing hormone. In the early 1960s, several investigators reported experimental results that were best explained by proposing the existence of substances that specifically stimulated the secretion of luteinizing hormone, and that were probably polypeptides, in crude aqueous extracts of hypothalamic tissues of various mammalian species (1-3). Preparations of LRF, active at 1 /Ag per dose in animal bioassays, were obtained by gel filtration and ion-exchange chromatography on carboxymethylcellulose (4), an observation that was confirmed by similar methods by several investigators (5, 6). In spite of the vagaries of the various bioassay methods available, several laboratories reported preparations of LRF of increased potency (5, 6). Several of these early publications led to contradictory statements regarding purification and separation of LH-releasing factor (LRF), from a follicle-stimulating hormone releasing factor (5, 7). Two laboratories independently reported the isolation of porcine LRF (8) and ovine LRF (9), both groups concluding that LRF from either species was a nonapeptide containing, on the basis of acid hydrolysis, 1 His, 1 Arg, 1 Ser, 1 Glu, 1 Pro, 2 Gly, 1 Leu, 1 Tyr. Earlier results with the pyrrolidone-carboxylylpeptidase prepared by Fellows and Mudge (10) had led us to conclude (11) that the Nterminal residue of LRF was Glu in its cyclized pyroglutamic (pGlu) form, as in the case of hypothalamic TRF, (pGluHis-Pro-NH2).While our own studies on the amino-acid sequence of ovine LRF were in progress, Matsuo et al. (12) reported that porcine LRF contai...

A method for preparing short-term enzymatically dispersed pituitary cell cultures is described. It has been used to assay hypothalamic releasing factors. The cells secrete TSH or LH and FSH in amounts directly related to the dose of TRF or LRF respectively. The minimal active doses of synthetic TRF and either synthetic or highly purified ovine LRF is ca. 10~1 0 M. Apparent affinity constants for TRF or LRF receptors are ca. 10~°M. Enhanced secretion rates of TSH or LH of 8-to 20-fold in response to maximal levels of TRF or LRF are routinely observed. The sensitivity of the cultured cells to TRF and LRF is maintained for more than 2 weeks in culture although the magnitude of the secretory response and the intracellular hormone content declines with time in culture. Chronic administration of TRF or LRF depletes the intracellular hormone levels while simultaneously increasing the total TSH or LH content in the culture (tissue and fluid). As has been observed with other in vitro systems, high medium potassium, prostaglandin E 2 and theophylline stimulate TSH secretion from the cultured cells. Furthermore, thyroid hormones which suppress the TSH secretion in response to TRF by the anterior pituitary in vivo and in other in vitro methods are similarly effective on these cells. The use of this technique has been applied to pituitary tissues of other species and types of cells, e.g., cells from Gj mouse thyrotropic tumors were shown to respond appropriately to TRF and thyroxine. The enzymatically dispersed cultured cells have been found to behave as would have been expected on the basis of experience with other in vitro methods while having significant advantages over those other procedures.

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Adrenocortical responsiveness to turning stress was examined in wild, reproductively‐active olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting (arribada) behavior. We hypothesized that the high sensitivity threshold (HST) observed in ovipositing sea turtles is associated with a diminished sensitivity of the hypothalamo‐pituitary‐adrenal (HPA) axis to stressful stimuli in arribada females. We tested this hypothesis by determining whether arribada females exhibited an increased activation threshold of the HPA axis to an imposed stressor (turning stress). Mean basal corticosterone (B) and glucose levels were below 1.0 ng/ml and 60 mg/dl, respectively. Basal B remained unchanged throughout a 24‐hr period in basking females. Most animals responded to turning stress with elevated mean B levels (up to 6.5 ng/ml after 6 hr) and no increase in circulating glucose. Nearly 50% of females (and none of the males) were refractory to the stimulation. Males exhibited the most rapid response, with B levels significantly elevated by 20 min over basal levels. Among females, arribada and solitary nesters exhibited a slower rate of response than basking, non‐nesting animals. These results demonstrate that olive ridleys exhibit stress‐induced changes in circulating B which are slower than those observed in most reptilian and in mammalian, avian, and piscine species. Furthermore, the presence of refractory females and the relatively slower increase in B in arribada and solitary nesters indicate a hyporesponsiveness of the HPA axis to turning stress in nesting olive ridleys. The hyporesponsiveness may be part of a mechanism to facilitate arribada nesting. J. Exp. Zool. 284:652–662, 1999. © 1999 Wiley‐Liss, Inc.