Corticotropin-releasing factor decreases plasma luteinizing hormone levels in female rats by inhibiting gonadotropin-releasing hormone release into hypophysial-portal circulation

To evaluate whether the hypothalamus is the site of action of CRF in inhibiting LH levels in female rats, we measured hypophysial-portal blood concentrations of immunoreactive GnRH (irGnRH) after the central injection of CRF. Ovine CRF (0.1, 1.0, 2.0, and 5.0 nmol) was injected intracerebroventricularly to intact rats on the afternoon of proestrus and in long term ovariectomized (OVX) rats in the presence or in absence of estradiol benzoate (OVX + EB). CRF injection decreased the amplitude of the proestrous irGnRH surge without affecting presurge levels. CRF (0.1 nmol) attenuated the afternoon irGnRH surge in OVX + EB rats; higher doses of CRF blocked this surge and decreased nonsurge irGnRH levels. No dose-related alterations of irGnRH levels were observed in OVX rats; only the highest dose of CRF was active. For comparison, plasma LH concentrations were measured after a single dose of CRF (2 nmol) in rats under the same experimental conditions. While CRF decreased LH concentrations in anesthetized proestrous and OVX + EB rats, it was inactive in OVX rats. In contrast, CRF injection in awake rats did decrease LH concentrations in all experimental conditions, suggesting that in OVX rats, the anesthetic (Saffan) used during portal blood collection affected CRF action on LH secretion. Indeed, the observation that the LH response to opiate receptor blockade with naloxone (2.5 mg/kg) in anesthetized OVX rats was different compared to that in awake rats suggested that the ineffectiveness of CRF to decrease irGnRH and LH in OVX anesthetized rats was related to the action of the anesthetic on the opioid system. The existence of a putative CRF-opioid interaction in the inhibitory control of LH secretion was supported by the effectiveness of naloxone to reverse the CRF-induced decrease in LH levels in EB-treated and untreated OVX rats. These results indicate that CRF attenuates LH secretion by a central action to inhibit irGnRH release into the hypophysial-portal circulation and that this action is independent of basal concentrations of irGnRH and/or LH. Moreover, the present results support the involvement of endogenous opioids in mediating the effect of CRF on LH secretion.     

Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

Crohn's disease and myasthenia gravis: a possible role for thymectomy.

A female patient with a three year history of Crohn's disease of the colon developed myasthenia gravis. Despite diversion of the faecal stream by an ileostomy, and total colectomy, the patient had continuing problems with perineal and perianal abscesses and fistulas. Her myasthenia gravis became unresponsive to anti-cholinergics so a thymectomy was performed. The perineal and perianal disease improved subsequently. This case supports the theory that functional disturbances of the thymus may have a role in the pathogenesis of inflammatory bowel disease.     

TP53 mutations in malignant and premalignant Barrett''s esophagus

In order to improve the efficacy of endoscopic surveillance of Barrett's esophagus, markers of neoplastic progression in addition to dysplasia are required. The aim of the present study was to assess TP53 mutational analysis as a method of identifying patients with Barrett's esophagus who are at greatest risk of adenocarcinoma, for whom endoscopic surveillance is most appropriate. TP53 mutational analysis was initially performed on premalignant and malignant tissue from 30 patients undergoing esophagectomy for adenocarcinoma, and on premalignant biopsies from 48 patients participating in a Barrett's surveillance program. Surveillance patients were followed up endoscopically and histologically for a median of 5 years following TP53 assessment. Mutational analysis was performed by single-strand conformation polymorphism analysis and direct DNA sequencing. TP53 mutations were detected in 10 of 30 esophageal adenocarcinomas, and were more common in well-differentiated carcinomas. An identical TP53 mutation was detected in carcinoma and adjacent dysplasia. Two patients with premalignant Barrett's esophagus had TP53 mutations and one of these patients developed adenocarcinoma on follow up whilst the other has not yet progressed beyond metaplasia. No patient without TP53 mutation developed high-grade dysplasia or adenocarcinoma. TP53 mutations are detected in 33% of esophageal adenocarcinomas and in 4% of premalignant Barrett's esophagus in patients undergoing endoscopic surveillance. TP53 mutation can be detected before the development of high-grade dysplasia or carcinoma, and may be useful in stratifying the risk of adenocarcinoma in patients with Barrett's esophagus.     

A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca²⁺-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Chemical synaptic transmission is the primary mode of cellular communication within the nervous system. The presynaptic piece of this process encompasses a remarkable set of sequential and highly regulated interactions between a host of proteins, synaptic vesicles (SV), the plasma membrane, and calcium ions (Ca²⁺). Fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane is driven by the assembly of the neuronal SNAREs SNAP-25 and Syntaxin 1 on the plasma membrane and Synaptobrevin-2/VAMP2 on the SV. The assembly process and its coupling to intracellular Ca²⁺ are choreographed by a deeply conserved group of proteins including Munc13, Munc18, Synaptotagmin 1, and Complexin (1–4). Together with the SNAREs, these proteins form the core of the fusion apparatus across all metazoan nervous systems (5–7).First identified in a landmark genetic screen for nervous system mutants in the nematode Caenorhabditis elegans, UNC-13 is the founding member of the highly conserved metazoan Unc13 secretory protein family that includes Unc13ABC in humans (Munc13-1/2/3 in mice) (8–10). Munc13-1/UNC-13 localizes to the presynaptic active zone and is implicated in numerous presynaptic functions including initiation of release site assembly, SV docking and priming, Ca²⁺- and lipid-dependent forms of short-term synaptic plasticity, opening and positioning Syntaxin 1 for SNARE assembly, and protecting SNARE complexes from disassembly by NSF/alpha-SNAP (3, 11–13). Loss of Munc13-1 orthologs in the nervous system almost entirely eliminates all forms of chemical synaptic transmission, establishing the Unc13 family as essential to this process (14–16). All UNC-13 orthologs contain a large Syntaxin-binding MUN domain flanked by a Ca²⁺- and lipid-binding C1-C2 module and an additional C2 domain on its C terminus referred to as C2C (5, 10, 17).The C-terminal end of UNC-13 is the least understood domain within the Unc13 protein family in terms of both structure and mechanism (18, 19). Recent work on the MUN and C2C domains of Munc13-1 both in vitro and in cultured hippocampal synapses supports the notion that the MUN-C2C region attaches Munc13-1 to SVs as a means of preparing SVs for fusion (20, 21), but several questions remain unresolved. Is the SV interaction mediated by direct membrane binding? Does the C2C domain itself bind to SVs or does the MUN domain serve this role? Does either domain provide cargo specificity as part of the priming process? Interestingly, the C-terminal end of the MUN domain of CAPS, another Unc13 family member, can bind dense-core vesicles (DCVs) although it lacks a C-terminal C2 domain (22). Moreover, the MUN domain without the C2C domain has also been demonstrated to bind liposomes through an interaction with Synaptobrevin 2 (23). These observations bring up several possibilities for interactions with the C terminus of Munc13 including direct MUN–membrane interactions, C2C–membrane interactions, or protein–protein interactions involving either or both domains. Other Unc13 family members possessing a MUN domain with a C-terminal C2 domain such as Unc13D/Munc13-4 and BAIAP3 have been proposed to tether specific cargo such as endosomes, secretory granules, and large DCVs (24, 25). How Unc13 proteins select among different cargos remains largely unanswered (24, 26, 27).Through behavioral, electrophysiological, biochemical, and genetic approaches, we uncover a deeply conserved C-terminal membrane-binding domain within Munc13-1/UNC-13 termed the Munc13 C-terminal (MCT) domain. This region, together with C2C and a neighboring N-terminal helix fold together into a stable membrane-binding protein domain in vitro, and loss of any part of this module in vivo impairs SV priming and nervous system function. Moreover, the C-terminal domain can be replaced by foreign domains that bind SVs but not the plasma membrane, demonstrating a role in SV interactions at the synapse. Phylogenetic protein sequence comparisons suggest that the ancestral Unc13/BAIAP3 homolog possessed a similar C-terminal domain prior to the emergence of metazoa, and subsequently, the UNC-13ABC subfamily domain evolved as an SV adaptor that plays a critical role in neurotransmission in all animals.     

Critical analysis of common canister programs: a review of cross-functional considerations and health system economics

Respiratory inhalers constitute a large percentage of hospital pharmacy expenditures. Metered-dose inhaler (MDI) canisters usually contain enough medication to last 2 to 4 weeks, while the average hospital stay for acute hospitalizations of respiratory illnesses is only 4–5 days. Hospital pharmacies are often unable to operationalize relabeling of inhalers at discharge to meet regulatory requirements. This dilemma produces drug wastage. The common canister (CC) approach is a method some hospitals implemented in an effort to minimize the costs associated with this issue. The CC program uses a shared inhaler, an individual one-way valve holding chamber, and a cleaning protocol. This approach has been the subject of considerable controversy. Proponents of the CC approach reported considerable cost savings to their institutions. Opponents of the CC approach are not convinced the benefits outweigh even a minimal risk of cross-contamination since adherence to protocols for hand washing and disinfection of the MDI device cannot be guaranteed to be 100% (pathogens from contaminated devices can enter the respiratory tract through inhalation). Other cost containment strategies, such as unit dose nebulizers, may be useful to realize similar reductions in pharmacy drug costs while minimizing the risks of nosocomial infections and their associated medical costs. The CC strategy may be appropriate for some hospital pharmacies that face budget constraints, but a full evaluation of the risks, benefits, and potential costs should guide those who make hospital policy decisions.