Corneal Changes and Strategies to Improve Survival of Hypomorphic Collagen VII-Deficient Mice for the Study of Ocular Dystrophic Epidermolysis Bullosa

Ophthalmic study of collagen CVII hypomorphic mice is uniquely challenging due to the strain’s published survival rate to weaning of 24%. Because chronic ocular fibrosis requires time to develop, optimizing the survival rate is of critical importance. In this study, standard husbandry practices were enhanced by the addition of sterilized diet and drug delivery gels, acidified water, irradiated food pellets, cellulose fiber bedding, minimal handling, removal of siblings within 2-3 wk from birth, and a preferred housing location. Survival rates per breeding cycle, sex, weight, and cause of early euthanasia were recorded and analyzed over 43 mo. Overall, 49% of mice survived to weaning and 76% of weaned mice survived to 20 wk of age. Corneal opacities were seen in 65% of mice by 20 wk, but only 10% of eyes showed the sustained opacification that was indicative of fibrosis. Corneal opacities occurred at the same rate as in humans with epidermolysis bullosa. 66% of the mice showed weight loss at 11 wk. Males required early euthanasia 4 times more often than did females. Euthanasia was required for urinary obstruction due to penile prolapse in 88% of males. With our enhanced care protocol, hypomorphic mice in our colony survived at twice the published rate. With this revised husbandry standard, experiments planned with termination endpoints of 14 wk for males and 17 wk for females are more likely to reach completion.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Uptake and biotransformation of pure commercial microcystin-LR versus microcystin-LR from a natural cyanobacterial bloom extract in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis>

Objectives

To evaluate the remediation efficiency of Mucor hiemalis by comparing media elimination, uptake, and biotransformation of microcystin-LR with exposure to pure toxin versus a crude bloom extract.

Results

With exposure to the extract, the elimination rate of microcystin-LR from the media, which was 0.28 ng MC-LR l^?1 h^?1, was significantly higher compared to that achieved with exposure to the pure toxin (0.16 ng MC-LR l^?1 h^?1) after 24 h. However, intracellular breakdown of microcystin-LR was significantly lower in the extract exposed pellets compared to the pure toxin treated fungal pellets over time. This coincided with reduced intracellular glutathione S-transferase activity with crude extract exposure which could be responsible for the detection of only the glutathione conjugate of microcystin-LR.

Conclusion

This paper signifies the importance of using laboratory exposure scenarios which resemble conditions in nature to fully understand and evaluate remediation efficiency. There is merit in using M. hiemalis for mycoremediation of cyanotoxins in surface waters.

     

The "ins" and "outs" of the high-affinity choline transporter CHT1

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.     

Hyperbaric oxygen prevents early death caused by experimental cerebral malaria

Background

Cerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis.

Methodology/Principal Findings

C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O₂, 3.0 ATA, 1–2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-α, IFN-γ and IL-10 mRNA levels and percentage of γδ and αβ CD4⁺ and CD8⁺ T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB) dysfunction and hypothermia.

Conclusions/Significance

The data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.     

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Introduction

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.

Methods

Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.

Results

Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.

Conclusions

The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.     

Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.