Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

Proteomic analysis of resting and thrombin‐stimulated platelets reveals the translocation and functional relevance of HIP‐55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co‐ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin‐stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS‐PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT‐ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip‐55. HIP‐55 is an SH3‐binding protein important in T‐cell receptor signalling. Further analysis of HIP‐55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin β3 upon platelet activation. Analysis of HIP‐55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP‐55 to be an important regulator of platelet function.     

Can splenocytes enhance pancreatic β-cell function and mass in 90% pancreatectomized rats fed a high fat diet?

AimsRecent studies have shown that splenocytes may act as a possible neogenic source with regard to β-cells in rodent diabetic models. Accordingly, we sought to determine whether splenocytes played an important role in promoting β-cell function and mass among type 2 diabetic rats with and without spleen.Main methodsWe randomly divided female 90% pancreatectomized (Px) Sprague Dawley rats into three groups: splenectomy (SPX), splenectomy plus the injection of male splenocytes (SPI), and no splenectomy (NSP). They were administered with 40 energy percent fat diets over the course of five weeks. At the end of the experimental period, insulin secretion capacity was measured by hyperglycemic clamp. At 6 h after BrdU⁺ injection, the pancreas was prepared with 4% paraformaldehyde in order to perform immunohistochemistry.Key findingsSPX increased and sustained serum glucose levels more than NSP and SPI during oral glucose tolerance testing. During hyperglycemic clamp, first and second phase insulin secretion decreased in the SPX rats while splenocyte injections counteracted this. Beta-cell mass in the SPX group was reduced more than among NSP and SPI. This was the result of a decrease in the number of small β-cell clusters in SPX, which is indicative of a decrease in β-cell neogenesis.SignificanceSplenocytes play an important role with regard to the neogenesis of β-cells in insulin deficient type 2 diabetic rats, although they are not critical for β-cell regeneration.     

Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus

SHP-1 is a Src homology 2 (SH2) domain-containing tyrosine phosphatase that plays an essential role in negative regulation of immune cell activity. We describe here a new model for regulation of SHP-1 involving phosphorylation of its C-terminal Ser591 by associated protein kinase Calpha. In human platelets, SHP-1 was found to constitutively associate with its substrate Vav1 and, through its SH2 domains, with protein kinase Calpha. Upon activation of either PAR1 or PAR4 thrombin receptors, the association between the three proteins was retained, and Vav1 became phosphorylated on tyrosine and SHP-1 became phosphorylated on Ser591. Phosphorylation of SHP-1 was mediated by protein kinase C and negatively regulated the activity of SHP-1 as demonstrated by a decrease in the in vitro ability of SHP-1 to dephosphorylate Vav1 on tyrosine. Protein kinase Calpha therefore critically and negatively regulates SHP-1 function, forming part of a mechanism to retain SHP-1 in a basal active state through interaction with its SH2 domains, and phosphorylating its C-terminal Ser591 upon cellular activation leading to inhibition of SHP-1 activity and an increase in the tyrosine phosphorylation status of its substrates.     

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function.     

Effectiveness of programme for reducing cardiovascular risk for men in one general practice.

OBJECTIVES--To assess the effectiveness of a programme for reducing cardiovascular risk in men in terms of clinical measurements and perceptions of patients. DESIGN--Collection of paired data on men attending well person clinics over three to five years. Questionnaire to determine changes in risk related habits. SETTING--Well person clinics in rural general practice with five partners in mid-Wales. SUBJECTS--The first 687 men seen in the clinic: analysable data obtained on 520. Initial age range 28-60 years. MAIN OUTCOME MEASURES--Analysis of serum cholesterol concentration (mmol/l) and blood pressure (mm Hg). Changes in diet, exercise, smoking, and drinking. RESULTS--Mean (SD) cholesterol concentration for all subjects increased from 5.8 (1.0) to 6.0 (1.0), p < 0.001. Overall percentage of ideal weight and mean systolic blood pressure also increased. Mean diastolic blood pressure was unchanged at 84 mm Hg. Professed dietary change, age, and number of visits all had significant effects on final cholesterol concentration. Those seen more often showed a significant decrease in cholesterol concentrations (in those seen more than twice mean (SD) initial value 6.7 (0.9) mmol/l v final value 6.5 (1.0) mmol/l, p < 0.001). Those over 45 years at start of study showed a significant decrease in diastolic blood pressure (mean (SD) initial value 89 (9) mm Hg v final value 86 (7) mm Hg, p < 0.001). CONCLUSIONS--These clinics are not effective in achieving a reduction in mean cholesterol concentration or blood pressure, despite evidence that the message of reduction in risk had been successfully transmitted. Success was achieved in older patients and those in whom a definite problem was identified. Identification of high risk groups and clinical case finding with appropriate long term follow up may be more cost effective in reducing cardiovascular risk than non-specific screening of the whole population.     

CH4 and N2O emissions from smallholder agricultural systems on tropical peatlands in Southeast Asia

There are limited data for greenhouse gas (GHG) emissions from smallholder agricultural systems in tropical peatlands, with data for non-CO₂ emissions from human-influenced tropical peatlands particularly scarce. The aim of this study was to quantify soil CH₄ and N₂O fluxes from smallholder agricultural systems on tropical peatlands in Southeast Asia and assess their environmental controls. The study was carried out in four regions in Malaysia and Indonesia. CH₄ and N₂O fluxes and environmental parameters were measured in cropland, oil palm plantation, tree plantation and forest. Annual CH₄ emissions (in kg CH₄ ha⁻¹ year⁻¹) were: 70.7 ± 29.5, 2.1 ± 1.2, 2.1 ± 0.6 and 6.2 ± 1.9 at the forest, tree plantation, oil palm and cropland land-use classes, respectively. Annual N₂O emissions (in kg N₂O ha⁻¹ year⁻¹) were: 6.5 ± 2.8, 3.2 ± 1.2, 21.9 ± 11.4 and 33.6 ± 7.3 in the same order as above, respectively. Annual CH₄ emissions were strongly determined by water table depth (WTD) and increased exponentially when annual WTD was above −25 cm. In contrast, annual N₂O emissions were strongly correlated with mean total dissolved nitrogen (TDN) in soil water, following a sigmoidal relationship, up to an apparent threshold of 10 mg N L⁻¹ beyond which TDN seemingly ceased to be limiting for N₂O production. The new emissions data for CH₄ and N₂O presented here should help to develop more robust country level ‘emission factors’ for the quantification of national GHG inventory reporting. The impact of TDN on N₂O emissions suggests that soil nutrient status strongly impacts emissions, and therefore, policies which reduce N-fertilisation inputs might contribute to emissions mitigation from agricultural peat landscapes. However, the most important policy intervention for reducing emissions is one that reduces the conversion of peat swamp forest to agriculture on peatlands in the first place.     

Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat

VIP-immunoreactive (IR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within microganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons.