Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Genome-wide screening reveals the emergence and divergence of RTK homologues in basal Metazoan <Emphasis Type="Italic">Hydra magnipapillata</Emphasis>

Receptor tyrosine kinases (RTKs) are key components of cell–cell signalling required for growth and development of multicellular organisms. It is therefore likely that the divergence of RTKs and associated components played a significant role in the evolution of multicellular organisms. We have carried out the present study in hydra, a diploblast, to investigate the divergence of RTKs after parazoa and before emergence of triploblast phyla. The domain-based screening using Hidden Markov Models (HMMs) for RTKs in Genomescan predicted gene models of the Hydra magnipapillata genome resulted in identification of 15 RTKs. These RTKs have been classified into eight families based on domain architecture and homology. Only 5 of these RTKs have been previously reported and a few of these have been partially characterized. A phylogeny-based analysis of these predicted RTKs revealed that seven subtype duplications occurred between ‘parazoan–eumetazoan split’ and ‘diploblast–triploblast split’ in animal phyla. These results suggest that most of the RTKs evolved before the radiata–bilateria divergence during animal evolution.     

Can anti-cyclic citrullinated peptide antibody-negative RA be subdivided into clinical subphenotypes?

Introduction  

Studies investigating genetic risk factors for susceptibility to rheumatoid arthritis (RA) studied anti-citrullinated peptide antibody (CCP)-positive RA more frequently than anti-CCP-negative RA. One of the reasons for this is the perception that anti-CCP-negative RA may include patients that fulfilled criteria for RA but belong to a wide range of diagnoses. We aimed to evaluate the validity of this notion and explored whether clinical subphenotypes can be discerned within anti-CCP-negative RA.     

Effect of Anionic Salt and Highly Fermentable Carbohydrate Supplementations on Urine pH and on Experimentally Induced Hypocalcaemia in Cows

The objective of this experiment was to determine the effect of dietary grain on calcium homeostasis. Six rumen-fistulated dairy cows with 3 or more previous lactations and no history of parturient paresis were randomly assigned to a sequence of diets in a crossover study with 4 periods of 10 days each. Dietary treatments were: A control ration consisting of wrap grass silage alone (1), the control ration supplemented with ammonium chloride and ammonium sulphate salt solution (2), control ration following a period with supplementation (3) and control ration supplemented with increasing amounts of barley from 4 to 10 kg/cow per day, expected to produce subclinical rumen acidosis (4). Daily intake of the diets was adjusted to 14 kg DM/cow per day. On day 11, the calcium-regulating mechanisms in cows were challenged until recumbency by a standardized intravenous EDTA infusion and cows were left to recover spontaneously. Anion supplementation and the feeding of highly fermentable carbohydrate lowered urine pH below 7.0 due to subclinical acidosis. During spontaneous recovery from EDTA induced hypocalcaemia, the cows more quickly regained a whole blood free calcium concentration of 1.00 mmol/L if they had most recently been supplemented with either anionic salts or with increasing amounts of barley, as compared to the basic ration. It is concluded that so-called slug-feeding or 'steaming up' with highly fermentable carbohydrates before parturition in milk fever susceptible cows enhanced calcium homeostasis similar to the effect seen in cows on anionic diets.     

Effect of nicotine on vasoconstrictor and vasodilator responses in human skin vasculature

Our objective was to test the hypothesis that acute exposure of human skin vasculature to nicotine may have deleterious effects on endothelial function. Vasoconstriction and vasorelaxation in isolated perfused human skin flaps (approximately 8 x 18 cm) derived from dermolipectomy specimens were assessed by studying changes in skin perfusion pressure measured by a pressure transducer, and skin perfusion was assessed by a dermofluorometry technique (n = 4 or 5). It was observed that nicotine (10(-7) M) amplified (P < 0.05) the norepinephrine (NE)-induced concentration-dependent (10(-7)-10(-5) M) increase in skin vasoconstriction compared with the control. This amplification effect of nicotine in NE-induced skin vasoconstriction was not blocked by the nicotine-receptor antagonist hexamethonium (10(-6) M) or the cyclooxygenase inhibitor indomethacin (10(-5) M). It was also observed that ACh and nitroglycerin (NTG) elicited a concentration-dependent (10(-8)-10(-5) M) vasorelaxation in skin flaps preconstricted with 8 x 10(-7) M of NE. The vasorelaxation induced by ACh was attenuated (P < 0.05) in the presence of nicotine (10(-7) M) compared with the control. However, skin vasorelaxation induced by NTG was not affected by nicotine (10(-7) M). ACh and NTG are known to induce endothelium-dependent and -independent vasorelaxation, respectively. The present findings were interpreted to indicate that acute exposure of human skin vasculature to nicotine was associated with 1) amplification of NE-induced skin vasoconstriction and 2) impairment of endothelium-dependent skin vasorelaxation. Cyclooxygenase products and nicotine receptors blocked by hexamethonium were not involved in the amplification of NE-induced skin vasoconstriction by nicotine. These findings may provide further insight into the pathogenesis of skin vasospasm in skin flap surgery and skin ischemic disease associated with cigarette smoking or use of smokeless tobacco.     

The vascularized pig fibula bone flap model: effect of segmental osteotomies and internal fixation on blood flow

The free fibular flap is the flap of choice for reconstruction of complex mandibular defects, although two or more osteotomies may be required to recreate the normal mandibular contour. The effect of these surgical manipulations on the fibula has not been adequately investigated. This study was designed to study the effect of multiple segmental osteotomies and internal fixation techniques on blood flow in the vascularized pig fibula bone flap model. The hindlimbs of 15 Yorkshire pigs were randomized into 1 of 5 groups (n = 6 fibulae per group) consisting of: (1) a nonoperated, in situ fibula; (2) an elevated fibula flap; (3) an elevated fibula flap with two segmental osteotomies; (4) an elevated fibula with two segmental closing osteotomies rigidly fixed with 2-mm miniplates; (5) an elevated fibula with two segmental closing osteotomies rigidly fixed with 2-mm lag screws. Total and gradient blood flow was measured in the bone and soft-tissue components of these flaps using the 15-microm radioactive microsphere technique. The creation of two segmental osteotomies in the vascularized pig fibula bone flap model resulted in a significant decrease (p<0.05) in the gradient blood flow in the segment of bone distal to the second osteotomy. Application of miniplates or lag screws across closing osteotomies resulted in a significant decrease (p<0.05) in total and gradient blood flow to the bone component of the fibulae, as compared with the elevated and osteotomized fibulae groups. An increase in blood flow suggesting a hyperemic response was noted in the bone and soft tissue in the elevated and osteotomized flap groups as compared with the in situ, nonoperated controls. This study established the validity of the pig fibula as a suitable model for investigating the pathophysiology of blood flow changes in the face of standard surgical maneuvers necessary for the restoration of mandibular form and function. The results demonstrated that the creation of multiple segmental osteotomies and the application of internal fixation significantly decreases (p<0.05) blood flow to the distal portion of the flap. The effects of segmental osteotomies and internal fixation on healing and growth of the pig fibula bone flap model are investigated in a separate study.     

A Model Based on Facilitated Passive Diffusion is Needed to Describe Root Water Entry through Aquaporins

Despite abundant evidence that water transfer from soil to xylem occurs along a pathway regulated by aquaporins (AQPs) water entry is still modeled using principles of ordinary passive diffusion. Problems with this model have been known for some time and include variable intrinsic properties of conductivity Lp, changing reflection coefficients, σ, and an inability to accurately resolve osmotic differentials between the soil and xylem. Here we propose a model of water entry based on principles of facilitated passive diffusion and following Michaelis-Menten formalism. If one accepts that water entry is controlled, at least in part, by AQPs, then a model of ordinary passive diffusion is precluded, as it does not allow for facilitation kinetics. By contrast, recognition of facilitated water entry through protein channels could explain shortcomings of ordinary passive diffusion, such as diurnal variability in conductivity which we have recently shown is directly correlated to diurnal changes in PsPIP2-1 mRNA levels in Pisum sativum.Key Words: aquaporins, root water entry, facilitated passive diffusion, simple passive diffusion, biophysical models     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.