The circulatory influence on development of age-related macular degeneration and hearing and equilibrium impairments

This study attempts to answer the question if any level of head and neck circulation takes a part in development of Age-Related Macular Degeneration (ARMD) and hearing and equilibrium impairments. Condition of large blood vessels was examined by Color-Doppler ultrasound, and carotid and ophthalmic arteries were included. The microcirculatory changes were examined directly by fundus photography and fluorescein angiography and indirectly testing hearing and equilibrium. The study group included 40 patients (21 females, 19 males) aging from 53 to 84 years with different stages of ARMD. The control group included 40 patients (18 females, 22 males) aging from 51 to 82 years without ARMD. Patients were inhabitants of Primorsko-Goranska County. There was no relationship between ARMD and condition of large blood vessels because significant stenosis of carotid arteries was found in 2 patients (5%) in study group and 3 patients (7.5%) in the control group (p > 0.05). On the contrary, we found correlation between ARMD and hearing (p = 0.0127) and equilibrium impairments (p = 0.0242). Fluorescein angiograms shows raised number of ischemic retinal capillaries in patients with ARMD (p = 0.0053). Results lead to conclusion that circulatory disorders on microcirculatory level take a great part in development of ARMD and hearing and equilibrium impairments in the elderly. The key is damage of sensory cells of the retina and inner ear caused by microcirculatory disorders. Interesting data was noticed that 9 patients with more serious ARMD on one side of head had greater hearing loss on the same side. If we find a new treatment for microcirculatory disorders, maybe we can treat both sensory impairments in earlier stage.     

Hetero-oligomerization of reggie-1/flotillin-2 and reggie-2/flotillin-1 is required for their endocytosis

Reggie-1/flotillin-2 and reggie-2/flotillin-1 are membrane raft associated proteins which have been implicated in growth factor signaling, phagocytosis, regulation of actin cytoskeleton and membrane trafficking. Membrane and raft association of reggies is mediated by myristoylation, palmitoylation and oligomerization. We have shown that upon EGF stimulation of cells, reggie-1 is tyrosine phosphorylated by Src kinase and endocytosed into late endosomes. Here we have analyzed the mechanism of the EGF-stimulated endocytosis of reggies in more detail and show that the Src-mediated phosphorylation of reggie-1 is not the driving force for endocytosis. However, hetero-oligomerization with reggie-2 is necessary for the translocation of reggie-1, which does not take place in the absence of reggie-2. In addition, the Y163F mutant of reggie-1, which is not capable of undergoing endocytosis, oligomerizes poorly with reggie-2. EGF stimulation results in changes in the size but not in the stoichiometry of the reggie hetero-oligomers, and reggie-1 oligomer size is decreased by knockdown of reggie-2. Based on our findings, we propose a model according to which reggie hetero-oligomers are dynamic, and changes in the size of the hetero-oligomers result in endocytosis of the complex from the plasma membrane.     

Variations in heart rate and rhythm of harbor seal pups during rehabilitation

Heart rate and rhythm is regulated by the autonomic nervous system, which matures during the first months of life. Little is known about heart rate and rhythm development and potential arrhythmias in seal pups during rehabilitation in seal centers. Using an iPhone ECG device, 1 min ECGs were obtained from harbor seal pups admitted to a seal rehabilitation facility. ECGs were taken from 55 seals after admission, 53 seals after 14 d, and 52 seals prior to release. From 24 seal pups additional ECGs were taken daily for the first week of rehabilitation. At admission sinus rhythm with a median heart rate of 148 complexes per minute was detected, prior to release sinus bradycardia or sinus arrhythmia with a median heart rate of 104 complexes minute was present. P wave morphology was highly variable and single supra‐ and ventricular premature complexes were recorded in individual animals. The first 14 d were characterized by highly variable heart rates and rhythms, including episodes of sinus tachycardia and 2nd degree atrioventricular blocks. The reduction in heart rates and development of a regular heart rhythm during rehabilitation suggest adaptation to the unfamiliar environment, resolution of disease, and/or maturation of the autonomic nervous system.     

Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross‐linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host‐induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae. When aphids were fed on transgenic barley expressing shp double‐stranded RNA (shp‐dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild‐type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2 weeks, respectively, on wild‐type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild‐type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S. avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.     

Cell apoptosis as assessed by M30 expression in keratoacanthoma and squamous cell carcinoma

Involution displayed by keratoacanthoma (KA) represents an important difference between KA and squamous cell carcinoma (SCC). It has been suggested that apoptosis plays a part in process of involution of KA. Altogether 150 specimens were included in this study, 30 cases of each; normal skin (NS), proliferative (pKA) and regressing keratoacanthoma (rKA), well differentiated (wdSCC) and poorly differentiated (pdSCC) squamous cell carcinoma. All samples were examined immunohistochemically for expression of M30 protein. A significantly lower number of M30 positive cells has been detected in NS as compared to skin tumors examined (p<0.001), except for rKA (p=0.057). The highest percentage of M30 positive cells was detected in pdSCC (p<0.001) as compared with all other examined groups. Keratinocytes of normal and changed epidermis expressing higher levels of M30 protein were predominately found in sun-exposed areas (chi2=14.93; p=0.060). There was an increasing trend of M30 protein expression with increasing age of the patient in NS and skin tumors examined. Majority of skin tumors with higher percentage of M30 positive cells tended to display higher Ki-67 expression. M30 expression was highly correlated with bak (r=0.811; p=0.048) and granzyme B expression in rKA (r=0.733; p=0.015). Cell apoptosis as assessed by M30 expression is, generally, increased in examined skin tumors and related to cell proliferation. Cell apoptosis mediated by bak and granzyme B expression could contribute to KA regression.     

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

3-phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1(-/-) ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1(-/-) ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.