Atomic Force Microscopy Imaging of the Human Trigeminal Ganglion

This paper describes an investigation of gangliocytes via imaging semithin sections of two human trigeminal ganglia with an atomic force microscope (AFM). Whereas semithin sections are usually employed for transmission electron microscopy, we adopted this special type of sample preparation for our AFM studies to extract topographical data from the gangliocyte itself and from the nucleus, the nucleolus, the crystal-arranged lipofuscin granules, and the cell-surrounding mantle cells; simultaneously we characterized the samples with error signal mode. This AFM-related technique revealed no information concerning friction force and elasticity due to the presence of the embedding material (epoxy), but it gave additional topographical contrast. These are the first images of the human trigeminal ganglion by AFM.     

Effect of YC-1, an NO-independent,superoxide-sensitive stimulator of soluble guanylyl cyclase,on smooth muscle responsiveness to nitrovasodilators

1. We studied the effects of 3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3′ : 5′ cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters.
2. YC-1 elicited a direct concentration-dependent activation of sGC (EC₅₀ 18.6±2.0 μM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing K_M and increasing V_max. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound.
3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive.
4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 μM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 μM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation.
5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O₂⁻) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O₂⁻ in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O₂⁻ at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1.
6. In conclusion, these results indicate that YC-1 is an NO-independent, O₂⁻-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.

     

The Genetics of Fetal Alcohol Spectrum Disorders

The term “fetal alcohol spectrum disorders” (FASD) defines the full range of ethanol (EtOH)‐induced birth defects. Numerous variables influence the phenotypic outcomes of embryonic EtOH exposure. Among these variables, genetics appears to play an important role, yet our understanding of the genetic predisposition to FASD is still in its infancy. We review the current literature that relates to the genetics of FASD susceptibility and gene–EtOH interactions. Where possible, we comment on potential mechanisms of reported gene–EtOH interactions. Early indications of genetic sensitivity to FASD came from human and animal studies using twins or inbred strains, respectively. These analyses prompted searches for susceptibility loci involved in EtOH metabolism and analyses of candidate loci, based on phenotypes observed in FASD. More recently, genetic screens in animal models have provided an additional insight into the genetics of FASD. Understanding FASD requires that we understand the many factors influencing phenotypic outcome following embryonic EtOH exposure. We are gaining ground on understanding some of the genetics behind FASD, yet much work remains to be carried out. Coordinated analyses using human patients and animal models are likely to be highly fruitful in uncovering the genetics behind FASD.     

Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging

Abstract: Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two‐dimensional (2‐D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun‐protected human foreskin samples pooled from ‘young’ and ‘old’ men. In addition, we analyzed these candidate gene products by 1‐D and 2‐D western blotting to obtain corroborative protein expression data, and by both real‐time PCR (RT‐PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post‐translationally regulated candidates such as phosphatidyl‐ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).     

Out-of-hours service in rural areas: An observational study of accessibility,attitudes and quality standards among general practitioners in Iceland

Objectives - To examine the access, workload, duties, commitments and quality standards of primary care physicians (GPs) resulting from out-of-hours service. Setting - All GPs (n=96) in rural Iceland. Main outcome measures - Answers to a postal survey. Results - The participation rate was 80%. The GPs estimated that in 97% of the cases they could be contacted within 5 minutes in an emergency. Under usual circumstances (weather conditions) and within a distance of 10 km, 70% of them could reach the patient within 30 minutes of receiving the call. In severe weather conditions, 50% of the GPs in smaller districts (650-6000 inhabitants) estimated that it could take up to 5 hours or more to reach the patient (which could happen once a year). In the least populated districts, 84% of the GPs had to be on call 14 days or more per month. Serious emergencies (involving special training such as cardiac resuscitation or tracheal intubation) were relatively rare, and GPs expressed the necessity for regular refresher courses in such fields. Conclusions - Modern telecommunication networks guarantee good access to out-of-hours service. The workload and on-call duties are great and do not comply with European Union (EU) recommendations regarding minimal rest time. If GPs in rural areas are to be expected to provide frontline health care, including in severe emergency situations, regular training courses are needed.