High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Role of the pseudorabies virus gI cytoplasmic domain in neuroinvasion, virulence, and posttranslational N-linked glycosylation

The glycoproteins I and E of pseudorabies virus are important mediators of cell-to-cell spread and virulence in all animal models tested. Although these two proteins form a complex with one another, ascribing any function to the individual proteins has been difficult. We have shown previously, using nonsense mutations, that the N-terminal ectodomain of the gE protein is sufficient for gE-mediated transsynaptic spread whereas the cytoplasmic domain of the protein is required for full expression of virulence. These same studies demonstrated that the cytoplasmic domain of gE is also required for endocytosis of the protein. In this report, we describe the construction of viruses with nonsense mutations in gI that allowed us to determine the contributions of the gI cytoplasmic domain to protein expression as well as virus neuroinvasion and virulence after infection of the rat eye. We also constructed double mutants with nonsense mutations in both gE and gI so that the contributions of both the gE and gI cytoplasmic domains could be determined. We observed that the gI cytoplasmic domain is required for efficient posttranslational modification of the gI protein. The gE cytoplasmic domain has no effect on gE posttranslational glycosylation. In addition, we found that infection of all gE-gI-dependent anterograde circuits projecting from the rat retina requires both ectodomains and at least one of the cytoplasmic domains of the proteins. The gI cytoplasmic domain promotes transsynaptic spread of virus better than the gE cytoplasmic domain. Interestingly, both gE and gI cytoplasmic tails are required for virulence; lack of either one or both results in an attenuated infection. These data suggest that gE and gI play differential roles in mediating directional neuroinvasion of the rat; however, the gE and gI cytoplasmic domains most likely function together to promote virulence.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Development of Standardized Insulin Treatment Protocols for Spontaneous Rodent Models of Type 1 Diabetes

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.Abbreviations: BBDP, BioBreeding diabetes-prone; BBDR, BioBreeding diabetes-resistant; NOD, nonobese diabetic; PZI, protamine zinc insulin; T1D, type 1 diabetes; VAF, viral-antibody–free; ZT, Zeitgeber timeClinical trials to prevent or reverse type 1 diabetes (T1D) are predicated on preclinical study data obtained from animal models of the disease to determine agents that exhibit efficacy and translational potential. However, according to findings published over the past several years (summarized in references 2, 17, and 31), not all preclinical T1D studies are created equal. Without a standardized screening process, the hundreds of candidate therapeutic agents in development cannot be evaluated critically for translational potential. One parameter that varies considerably from report to report in T1D reversal studies is the insulin treatment provided to diabetic NOD mice. To address the need for standardized preclinical screening of new therapeutics, the National Institute for Diabetes and Digestive and Kidney Diseases has developed the Type 1 Diabetes Preclinical Testing Program.^2,35 Under this program, a central contract testing facility (Biomedical Research Models) bridged the gap between discovery of potential therapeutics and clinical testing for efficacy in prevention or reversal of T1D. Using 2 of the best characterized models of T1D, the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse, we sought to develop standardized protocols for the treatment of diabetes with insulin to provide the best glycemic control throughout the fed and nonfed states. We began by housing these models in a viral-antibody–free (VAF) barrier facility, we created study designs approved by a scientific advisory board consisting of leaders in the field, and we performed studies by using standard operation procedures.The standard of care in patients with T1D is to attempt to maintain near-normal glucose levels, by providing exogenous insulin therapy several times daily via injection or pump after rigorous monitoring of glycemic levels and by appropriately coordinating insulin dosing with food intake. Current blood glucose control in diabetic rodent models focuses on maintaining the diabetic animal in a state of moderate hyperglycemia, with normal weight gain in the absence of severe ketonuria. This state is achieved by once-daily injections of titrated insulin doses or by implantation of continuous release insulin pellets;³⁸ however, insulin types and methods can vary widely between institutions and laboratories, yielding a wide range of glycemic control. Therefore there is marked difference between the stringent glycemic control targeted by humans with diabetes as compared with the relatively loose glycemic control afforded to rodents with diabetes. Despite the many physiologic differences between humans and rodents, glycemic control potentially can be addressed by making insulin treatment in rodents more comparable in terms of glycemic control to what is achieved currently in humans, especially given that patients with T1D will continue to administer insulin during treatment with therapeutic agents (for example, antiCD3).¹¹ The lessening of the frequency, duration, and severity of hyperglycemic events is anticipated to provide the best chance for β cells to rest (function properly) while interventions are tested.²¹ Ideally, for these studies, animals should receive sufficient insulin to maintain glycemic levels close to the normal range in control nondiabetic animals.For these studies, we focused on the 2 most widely used spontaneous rodent models of T1D: the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse.^1,12 The BBDP strain originated from a colony of outbred Wistar rats that developed spontaneous diabetes at the BioBreeding Laboratories in the 1970s. In the 1980s, the strain was acquired by the University of Massachusetts Medical School. During inbreeding, the BioBreeding diabetes- resistant (BBDR) control strain was established. Both strains are maintained at our facility and represent the most fully inbred (more than 110 generations) and characterized colonies available. BBDP rats develop T1D at 50 to 90 d of age at a frequency of approximately 85% to 90%, with equal frequency in male and female rats; the disease in BBDP rats results from autoimmune insulitis that is mediated primarily by CD4⁺ and CD8⁺ T cells and the development of autoantibodies to islet antigen. This insulitis is similar to that in human patients.¹⁸ Insulin therapy is required shortly after onset of hyperglycemia or death will occur due to ketoacidosis.¹⁹ The Gimap5 mutation in BBDP rats results in a T-cell lymphopenia and is necessary for development of T1D in BBDP rats (along with expression of a MHC class II RT1 B/D^u allele); adoptive transfer of splenocytes or regulatory T cells from BBDR rats before 35 d of age prevents the onset of diabetes in BBDP rats.^9,28,38 Alternatively, depletion of regulatory T cells from BBDR rats (which are nonlymphopenic) induces T1D in that strain.The NOD mouse strain originated from selective inbreeding of the Cataract Shionogi mouse strain and was imported from Japan to The Joslin Diabetes Center in 1984. NOD mice are now the most widely used preclinical model of T1D, in part due to the availability of genetic analysis and manipulation as well as the wide array of reagents available for mechanistic studies. The most commonly cited source for NOD mice is The Jackson Laboratory (Bar Harbor, ME), where female NOD mice develop disease at a frequency of 65% to 100% by 30 wk of age, whereas male NOD mice develop disease at a frequency of 35% to 85% (inbred for more than 83 generations). The incidence can vary from year to year³⁴ and from facility to facility depending on several factors, the most important being housing conditions.^15,26 The incidence of T1D in female NOD mice at our VAF barrier facility has been 65% to 80% over the past 3 y; this frequency can be far lower in nonVAF facilities. Diabetic NOD mice exhibit mild ketoacidosis, which allows them to survive for as long as several weeks after the onset of hyperglycemia without supportive insulin treatment. NOD mice also present with insulin resistance and a distinct stage of insulitis, referred to as peri-insulitis, that is not found in either human T1D or in diabetic BBDP rats.^5,18 Although both NOD mouse and BBDP rat models of T1D have particular advantages and disadvantages, a prudent path of drug development would include the examination of the therapeutic efficacy of novel agents in both models.^2,31To standardize and improve current testing protocols, we developed insulin treatment regimens that maintain blood glucose levels near normal levels throughout day and night activities over prolonged periods, as would be expected to occur in interventional clinical trials. We show here that whereas 2 daily injections of insulin to diabetic BBDP rats were sufficient to achieve our goal, diabetic NOD mice required continuous delivery of insulin through the implantation of osmotic pumps.     

EFFECTS OF HUMAN ACTIVITIES ON STRUCTURE AND COMPOSITION OF WOODY SPECIES OF THE NOKREK BIOSPHERE RESERVE OF MEGHALAYA,NORTHEAST INDIA

Aims Our study was conducted in the Nokrek Biosphere Reserve (NBR) in the Garo hills districts of Meghalaya, Northeast India. Our aim was to assess the effects of human activities on plant diversity,population structure and regeneration.Methods We selected a representative 1.2 hm2 stand in both the core and buffer zones of NBR. Structure and composition were determined by randomly sampling square quadrats, population structure was assessed by determining age structure, and regeneration was assessed by measuring densities of seedling, sapling and adult trees.Important findings More woody species were recorded from the core zone than the buffer zone (87 vs. 81 species), and there were a large number of tropical, temperate, and Sino-Himalayan, Burma-Malaysian and Malayan elements, primitive families and primitive genera. The trees were distributed in three distinct strata,canopy, subcanopy and sapling. Subcanopy and sapling layers had the highest species richness (81％ -88％ ). Lauraceae and Euphorbiaceae were the dominant families in terms of the number of species, and a large number of families were represented by single species. Most woody species (57 ％ - 79 ％ ) were contagiously distributed and had low frequency ( ＜ 20％ ). Although stand density was high in the buffer zone, its basal area was low compared to the stand in the core zone. Low similarity and high β-diversity indicate marked differences in species composition of the stands. Shannon diversity index was high in both the stands, while Simpson dominance index was low. The diameter-class distribution for dominant species revealed that the most had a large number of young individuals in their populations. Preponderance of tree seedlings, followed by a steep decline in population density of saplings and adult trees, indicated that the seedling to sapling stage was the most critical in the life cycle of the tree populations. Most species (42 ％ - 48 ％ ) had no regeneration,25 ％ - 35 ％ had good/fair regeneration, and the rest had poor regeneration or reoccurred as immigrants.     

Distinctive modulation of inflammatory and metabolic parameters in relation to zinc nutritional status in adult overweight/obese subjects

Overweight and obesity are associated with low grade of inflammation and chronic inflammatory response characterized by abnormal production and activation of some pro-inflammatory signalling pathways. Taking into account that obesity is the direct result of an imbalance between energy intake and energy expenditure, the nutritional factors in the diet, with particular focus on zinc, may play a pivotal role in the development of obesity-associated comorbidities. Considering the potential interactions among zinc nutritional status, inflammation, overweight/obesity and insulin secretion, the aim of the present work was to clarify the influence of zinc dietary intake on some metabolic, inflammatory and zinc status parameters in adult overweight/obese subjects. We found a close interrelationship between nutritional zinc and obesity. In particular, subjects with a lower zinc dietary intake display a deeper inflammatory status, general impairment of the zinc status, an altered lipid profile and increased insulin production with respect to obese subjects with normal zinc dietary intake. Moreover, in the presence of low dietary zinc intake, the obese subjects are less capable to respond to oxidative stress and to inflammation leading to the development of obesity or to a worsening of already preexisting obesity status. In conclusion, a possible zinc supplementation in obese subjects with a deeper inflammatory status and more altered zinc profile may be suggested in order to limit or reduce the inflammation, taking also into account that zinc supplementation normalizes “inflammaging” as well as zinc profile leading to a correct intra- and extracellular zinc homeostasis.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.