The use of phenyl-Sepharose for the affinity purification of proteinases

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effective retained by the media. Purification factors in the range of 2.9–60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts

Post-ischaemic immunological response in the brain: targeting microglia in ischaemic stroke therapy

Microglia, the major endogenous immune cells of the central nervous system, mediate critical degenerative and regenerative responses in ischaemic stroke. Microglia become "activated", proliferating, and undergoing changes in morphology, gene and protein expression over days and weeks post-ischaemia, with deleterious and beneficial effects. Pro-inflammatory microglia (commonly referred to as M1) exacerbate secondary neuronal injury through the release of reactive oxygen species, cytokines and proteases. In contrast, microglia may facilitate neuronal recovery via tissue and vascular remodelling, through the secretion of anti-inflammatory cytokines and growth factors (a profile often termed M2). This M1/M2 nomenclature does not fully account for the microglial heterogeneity in the ischaemic brain, with some simultaneous expression of both M1 and M2 markers at the single-cell level. Understanding and regulating microglial activation status, reducing detrimental and promoting repair behaviours, present the potential for therapeutic intervention, and open a longer window of opportunity than offered by acute neuroprotective strategies. Pharmacological modulation of microglial activation status to promote anti-inflammatory gene expression can increase neurogenesis and improve functional recovery post-stroke, based on promising preclinical data. Cell-based therapies, using preconditioned microglia, are of interest as a method of therapeutic modulation of the post-ischaemic inflammatory response. Currently, there are no clinically-approved pharmacological options targeting post-ischaemic inflammation. A major developmental challenge for clinical translation will be the selective suppression of the deleterious effects of microglial activity after stroke whilst retaining (or enhancing) the neurovascular repair and remodelling responses of microglia

Digestion of Protein in Premature and Term Infants.

Premature birth rates and premature infant morbidity remain discouragingly high. Improving nourishment for these infants is the key for accelerating their development and decreasing disease risk. Dietary protein is essential for growth and development of infants. Studies on protein nourishment for premature infants have focused on protein requirements for catch-up growth, nitrogen balance, and digestive protease concentrations and activities. However, little is known about the processes and products of protein digestion in the premature infant. This review briefly summarizes the protein requirements of term and preterm infants, and the protein content of milk from women delivering preterm and at term. An in-depth review is presented of the current knowledge of term and preterm infant dietary protein digestion, including human milk protease and anti-protease concentrations; neonatal intestinal pH, and enzyme activities and concentrations; and protein fermentation by intestinal bacteria. The advantages and disadvantages of incomplete protein digestion as well as factors that increase resistance to proteolysis of particular proteins are discussed. In order to better understand protein digestion in preterm and term infants, future studies should examine protein and peptide fragment products of digestion in saliva, gastric, intestinal and fecal samples, as well as the effects of the gut micro biome on protein degradation. The confluence of new mass spectrometry technology and new bioinformatics programs will now allow thorough identification of the array of peptides produced in the infant as they are digested

Microglia in Alzheimer's disease: a role for ion channels

Alzheimer's disease is the most common form of dementia, it is estimated to affect over 40 million people worldwide. Classically, the disease has been characterized by the neuropathological hallmarks of aggregated extracellular amyloid-β and intracellular paired helical filaments of hyperphosphorylated tau. A wealth of evidence indicates a pivotal role for the innate immune system, such as microglia, and inflammation in the pathology of Alzheimer's disease. The over production and aggregation of Alzheimer's associated proteins results in chronic inflammation and disrupts microglial clearance of these depositions. Despite being non-excitable, microglia express a diverse array of ion channels which shape their physiological functions. In support of this, there is a growing body of evidence pointing to the involvement of microglial ion channels contributing to neurodegenerative diseases such as Alzheimer's disease. In this review, we discuss the evidence for an array of microglia ion channels and their importance in modulating microglial homeostasis and how this process could be disrupted in Alzheimer's disease. One promising avenue for assessing the role that microglia play in the initiation and progression of Alzheimer's disease is through using induced pluripotent stem cell derived microglia. Here, we examine what is already understood in terms of the molecular underpinnings of inflammation in Alzheimer's disease, and the utility that inducible pluripotent stem cell derived microglia may have to advance this knowledge. We outline the variability that occurs between the use of animal and human models with regards to the importance of microglial ion channels in generating a relevant functional model of brain inflammation. Overcoming these hurdles will be pivotal in order to develop new drug targets and progress our understanding of the pathological mechanisms involved in Alzheimer's disease

Nd:YLF laser for airborne/spaceborne laser ranging

In order to meet the need for light weight, long lifetime, efficient, short pulse lasers, a diode-pumped, Nd:YLF oscillator and regenerative amplifier is being developed. The anticipated output is 20 mJ per 10 picosecond pulse, running at a repetition rate of 40 Hz. The fundamental wavelength is at 1047 nm. The oscillator is pumped by a single laser diode bar and mode locked using an electro-optic, intra-cavity phase modulator. The output from the oscillator is injected as a seed into the regenerative amplifier. The regenerative amplifier laser crystal is optically pumped by two 60W quasi-cw laser diode bars. Each diode is collimated using a custom designed micro-lens bar. The injected 10 ps pulse from the oscillator is kept circulating within the regenerative amplifier until this nanojoule level seed pulse is amplified to 2-3 millijoules. At this point the pulse is ejected and sent on to a more standard single pass amplifier where the energy is boosted to 20 mJ. The footprint of the entire laser (oscillator-regenerative amplifier-amplifier) will fit on a 3 by 4 ft. optical pallet

Ventilator Dependency in ALS: Management, Disease Progression, and Issues of Coping

The natural progression of amyotrophic lateral sclerosis (ALS) leads to respiratory fail ure and death. Artificial ventilation can prolong the course, leading to extreme degrees of weakness and dependence. Little specific information is available to counsel ALS patients about making the decision for artificial ventilation. In order to gain more infor mation, we visited four ventilator-dependent ALS patients and their primary caregivers. We determined the neurologic state and level of function of the patients and inter viewed their primary caregivers to assess medical care and management needs (both social and financial) and how they were being met. We also administrated question naires to assess the psychological well-being of both patient and primary caregiver and how the relationship between the patient and primary caregiver changed under these circumstances. Key Words: ALS—Artificial ventilation—Disease progression.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68609/2/10.1177_154596839601000306.pd

Heat flow measurements on a hydrothermally-active, slow-spreading ridge: The Escanaba Trough

Maximum heat flow measurements at three locations in the sediment-filled Escanaba Trough of the Gorda ridge exceed 1200 mW/m². At other ridge crests with thick sediment cover, heat flow values of this magnitude are accompanied by high temperature hydrothermal vent activity and massive sulfide deposition. A dredge haul from the southernmost high heat flow location recovered pyrrhotite, thereby confirming the presence of recent high temperature venting

Effects of Stocking Density on Steer Performance and Carcass Characteristics in Bedded Hoop Barns

Use of bedded hoop barns for feeding cattle has grown in part due to increased regulations regarding open feedlot runoff. In 2010, ISU estimated almost 700 hoop barns in Iowa used for beef cattle and more than 80% were used for cattle feeding. Work in Iowa also has documented that cattle confined in a bedded hoop barn perform similarly to cattle fed in an open feedlot with shelter. The work was done with a stocking density of 50 sq ft per steer in the bedded hoop barn. A hoop barn is a more expensive facility system compared with open lot configurations. Fixed costs per steer (facilities) are partially determined by stocking density. The objective of this study was to determine the effects of increased stocking density on performance and carcass characteristics of steers fed in bedded hoop and bedded open front facilities. The trials were conducted in 2008 to 2011 at the ISU Armstrong Research Farm, Lewis, IA. The hoop barn was stocked with 40, 45 and 50 head per pen resulting in 50, 45 and 40 sq ft per steer, respectively. There was one stocking density per housing type per trial. The diet fed was 45.0% dry corn, 14.8% ground hay, 36.8% modified distillers grains and 3.4% supplement on an as-fed basis. The total diet was approximately 69% dry matter. Cattle growth rate (ADG), feed intake (DMI), feed efficiency (F/G) and mean mud score did not differ based on stocking density (P\u3e0.05). However, the cattle given more square feet numerically had greater feed intake and were more efficient (about 4-5%) than the more densely stocked cattle. This study may not have had enough replications to detect statistically significant differences. Also the mean carcass characteristics (fat cover, rib eye area, marbling score, quality grade and yield grade) did not differ by stocking density (P\u3e0.05). When comparing seasons only, steers fed in summer tended to have heavier market liveweight, heavier carcass weight, required less feed per liveweight and less marbling compared with steers fed in winter (P≤0.10). Also, the summer-fed steers grew faster and gained more liveweight than the winter-fed steers (P\u3c0.005). These results suggest that this study may not have reached the maximum stocking density for feeding beef cattle in a bedded hoop barn. In other words, market cattle can probably be stocked at less square footage per steer than the 40 sq ft per steer used in this study. Observations of farmers with hoop barns suggest that 37 or 35 sq ft per steer may be feasible. Several factors may affect stocking density including genetics or frame size of the cattle and may interact with season. Also, it should be noted that as the density of cattle increases, more management is required, i.e., more bedding, more bunk space, more waterer space and observing individual cattle for health issues becomes more difficult