Prevalence Rates of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae) in Host-Seeking Ixodes scapularis (Acari: Ixodidae) from Pennsylvania.

The etiological agents responsible for Lyme disease (Borrelia burgdorferi), human granulocytic anaplasmosis (Anaplasma phagocytophilum), and babesiosis (Babesia microti) are primarily transmitted by the blacklegged tick, Ixodes scapularis Say. Despite Pennsylvania having in recent years reported the highest number of Lyme disease cases in the United States, relatively little is known regarding the geographic distribution of the vector and its pathogens in the state. Previous attempts at climate-based predictive modeling of I. scapularis occurrence have not coincided with the high human incidence rates in parts of the state. To elucidate the distribution and pathogen infection rates of I. scapularis, we collected and tested 1,855 adult ticks statewide from 2012 to 2014. The presence of I. scapularis and B. burgdorferi was confirmed from all 67 Pennsylvania counties. Analyses were performed on 1,363 ticks collected in the fall of 2013 to avoid temporal bias across years. Infection rates were highest for B. burgdorferi (47.4%), followed by Ba. microti (3.5%) and A. phagocytophilum (3.3%). Coinfections included B. burgdorferi+Ba. microti (2.0%), B. burgdorferi+A. phagocytophilum (1.5%) and one tick positive for A. phagocytophilum+Ba. microti. Infection rates for B. burgdorferi were lower in the western region of the state. Our findings substantiate that Lyme disease risk is high throughout Pennsylvania.

Photoperiod-dependent regulation of carboxypeptidase E affects the selective processing of neuropeptides in the seasonal Siberian hamster (Phodopus sungorus).

The production of bioactive peptides from biologically inactive precursors involves extensive post-translational processing, including enzymatic cleavage by proteolytic peptidases. Endoproteolytic prohormone-convertases initially cleave the precursors of many neuropeptides at specific amino acid sequences to generate intermediates with basic amino acid extensions on their C-termini. Subsequently, the related exopeptidases, carboxypeptidases D and E (CPD and CPE), are responsible for removing these amino acids before the peptides achieve biological activity. We investigated the effect of photoperiod on the processing of the neuropeptide precursor pro-opiomelanocortin (POMC) and its derived neuropeptides, α-melanocyte-stimulating hormone (MSH) and ß-endorphin (END), within the hypothalamus of the seasonal Siberian hamster (Phodopus sungorus). We thus compared hypothalamic distribution of CPD, CPE, α-MSH and ß-END using immunohistochemistry and measured the enzyme activity of CPE and concentrations of C-terminally cleaved α-MSH in short-day (SD; 8 : 16 h light/dark) and long-day (LD; 16 : 8 h light/dark) acclimatised hamsters. Increased immunoreactivity (-IR) of CPE, as well as higher CPE activity, was observed in SD. This increase was accompanied by more ß-END-IR cells and substantially higher levels of C- terminally cleaved α-MSH, as determined by radioimmunoassay. Our results suggest that exoproteolytic cleavage of POMC-derived neuropeptides is tightly regulated by photoperiod in the Siberian hamster. Higher levels of biological active α-MSH- and ß-END in SD are consistent with the hypothesis that post-translational processing is a key event in the regulation of seasonal energy balance.

Marsupial uncoupling protein 1 sheds light on the evolution of mammalian nonshivering thermogenesis.

Brown adipose tissue expressing uncoupling protein 1 (UCP1) is responsible for adaptive nonshivering thermogenesis giving eutherian mammals crucial advantage to survive the cold. The emergence of this thermogenic organ during mammalian evolution remained unknown as the identification of UCP1 in marsupials failed so far. Here, we unequivocally identify the marsupial UCP1 ortholog in a genomic library of Monodelphis domestica. In South American and Australian marsupials, UCP1 is exclusively expressed in distinct adipose tissue sites and appears to be recruited by cold exposure in the smallest species under investigation (Sminthopsis crassicaudata). Our data suggest that an archetypal brown adipose tissue was present at least 150 million yr ago allowing early mammals to produce endogenous heat in the cold, without dependence on shivering and locomotor activity.

PC1/3 and PC2 gene expression and post-translational endoproteolytic pro-opiomelanocortin processing is regulated by photoperiod in the seasonal Siberian hamster (Phodopus sungorus).

A remarkable feature of the seasonal adaptation displayed by the Siberian hamster (Phodopus sungorus) is the ability to decrease food intake and body weight (by up to 40%) in response to shortening photoperiod. The regulating neuroendocrine systems involved in this adaptation and their neuroanatomical and molecular bases are poorly understood. We investigated the effect of photoperiod on the expression of prohormone convertases 1 (PC1/3) and 2 (PC2) and the endoproteolytic processing of the neuropeptide precursor pro-opiomelanocortin (POMC) within key energy balance regulating centres of the hypothalamus. We compared mRNA levels and protein distribution of PC1/3, PC2, POMC, adrenocorticotrophic hormone (ACTH), alpha-melanocyte-stimulating hormone (MSH), beta-endorphin and orexin-A in selected hypothalamic areas of long day (LD, 16:8 h light:dark), short day (SD, 8:16 h light:dark) and natural-day (ND, photoperiod depending on time of the year) acclimated Siberian hamsters. The gene expression of PC2 was significantly higher within the arcuate nucleus (ARC, P < 0.01) in SD and in ND (versus LD), and is reflected in the day length profile between October and April in the latter. PC1/3 gene expression in the ARC and lateral hypothalamus was higher in ND but not in SD compared to the respective LD controls. The immunoreactivity of PC1/3 cleaved neuropeptide ACTH in the ARC and PC1/3-colocalised orexin-A in the lateral hypothalamus were not affected by photoperiod changes. However, increased levels of PC2 mRNA and protein were associated with higher abundance of the mature neuropeptides alpha-MSH and beta-endorphin (P < 0.01) in SD. This study provides a possible explanation for previous paradoxical findings showing lower food intake in SD associated with decreased POMC mRNA levels. Our results suggest that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by post-translational processing mediated by the prohormone convertases PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors.

Circulating ghrelin levels and central ghrelin receptor expression are elevated in response to food deprivation in a seasonal mammal (Phodopus sungorus).

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). However, the functional interaction of ligand and receptor is not very well understood. We demonstrate that GHSR mRNA is up-regulated after food deprivation (48 h) in the hypothalamic arcuate nucleus and ventromedial nucleus of the seasonal Siberian hamster, Phodopus sungorus. This increase is accompanied by a two-fold elevation of circulating ghrelin concentration. Chronic changes in feeding state imposed by food restriction over a period of 12 weeks during long day-length induced increased GHSR gene expression, whereas food restriction for 6 weeks had no effect. Phodopus sungorus reveals remarkable seasonal changes in body weight, fat mass and circulating leptin levels. Ghrelin is generally regarded as having opposing effects on appetite and body weight with respect to those exhibited by leptin. However, our study revealed that seasonal adaptations were not accompanied by changes in either GHSR gene expression or circulating ghrelin concentration. Therefore, we suggest that ghrelin only plays a minor role in modulating long-term seasonal body weight cycles. Our findings imply that ghrelin predominantly acts as a short-term regulator of feeding.

Plasmodium chabaudi-infected erythrocytes: Differential expression of trophozoite proteins in host cell plasma membranes.

Neoproteins were compared in host cell plasma membranes isolated as ghosts from erythrocytes infected with early and late trophozoites of Plasmodium chabaudi. Ghosts isolated from early trophozoites-infected erythrocytes (= et-ghosts) contain seven neoproteins with apparent molecular masses of about 154, 145, 90, 72, 67, 52 and 33 kDa, respectively. Two additional neoproteins with masses of about 42 and 38 kDa are detected in ghosts isolated from late trophozoites-infected erythrocytes (= lt-ghosts). All neoproteins, except the 154, 145 and 38 kDa-proteins, can be metabolically labeled with both [(14)C]isoleucine and [(35)S]methionine. The 90 kDa-protein is predominantly labeled in et-ghosts, while the 42 kDa-protein is exclusively labeled in lt-ghosts. Both proteins, however, contain only minor radioactivity in the corresponding parasites. Moreover, the pI of the 90 kDa-neoprotein shifts from about 4.5 in et-ghosts to about 5.6 in lt-ghosts as detected by 2 D-gel electrophoresis and western blotting using a 90 kDa-protein specific monoclonal antibody. The data indicate a posttranslational modification of the 90 kDa-neoprotein and a sequential expression of the 90 kDa- and 42 kDa-neoproteins in host cell plasma membranes during intraerythrocytic maturation of trophozoites.

Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with Plasmodium chabaudi.

Erythrocytes infected with the malaria parasite Plasmodium chabaudi contain the neo-protein Pc90 in their plasma membrane. We investigate origin, membrane disposition, and intraerythrocytic traffic of this Pc90. Metabolic labeling of P.-infected erythrocytes, combined with cell fractionation as well as Western blot analysis and immunoprecipitation using a Pc90-recognizing monoclonal antibody, show that Pc90 is synthesized by early to mid trophozoites and is transported without any apparent processing steps to the erythrocyte membrane. Based upon the inaccessibility of Pc90 from the outside in intact erythrocytes and the water solubility of membrane-associated Pc90, it is concluded that Pc90 is localized on the cytoplasmic face of the host erythrocyte membrane. Immunoelectron microscopy using a Pc90-specific monoclonal antibody and the occurrence of soluble Pc90 in host cell cytosol indicate that the Pc90 is transported in both a 'vesicle-bound' and a 'free' form through the erythrocyte cytoplasm.

Plasmodium chabaudi malaria: protective immunization with surface membranes of infected erythrocytes.

Plasmodium chabaudi-susceptible NMRI and B10.A mice were vaccinated with host cell plasma membranes isolated from P. chabaudi-infected erythrocytes. Most of the mice were protected from the lethal consequences of challenge with the homologous parasite, although protection was unassociated with a reduction in the course or peak of parasitemia. Vaccination also induced the production of antibodies against Pc90, which is the immunodominant protein expressed by parasites in host cell plasma membranes.

Resistance to Plasmodium chabaudi in B10 mice: influence of the H-2 complex and testosterone.

Resistance to Plasmodium chabaudi has been examined in different inbred mouse strains bearing identical H-2 haplotypes on different genetic backgrounds as well as in H-2-congenic mouse strains on B10 background. Resistance is expressed in terms of percent survival after a challenge with 10(6) P. chabaudi-infected erythrocytes. We can show that murine resistance to P. chabaudi is under complex polygenic control involving a non-H-2 gene(s) as well as genes in both I-A and I-E subregions of the H-2 complex. Our data indicate in particular that malaria protective antigens can be presented in context with I-Ab molecules but not in context with I-Ak molecules. Resistance controlled by I-Ab does not become apparent when I-Ek molecules are coincidentally expressed. Moreover, testosterone abrogates I-Ab-controlled resistance to P. chabaudi.

Cryptic disposition of antigenic parasite proteins in plasma membranes of erythrocytes infected with Plasmodium chabaudi.

Plasma membranes of Plasmodium chabaudi-infected erythrocytes contain seven major neoproteins with apparent molecular masses of 154, 145, 90, 72, 67, 52, and 33 kDa, respectively. These neoproteins, with the exception of the two larger ones, can be metabolically labelled with [14C]isoleucine. The seven neoproteins are antigenic as revealed by Western blotting using hyperimmune sera obtained from two different mouse strains. None of the parasite proteins is accessible from the outside in intact P. chabaudi-infected erythrocytes as determined by lactoperoxidase-mediated radioiodination, indirect immune fluorescence microscopy, or post-embedding immunoelectron microscopy. These methods, however, identify parasite proteins in host cell plasma membranes when the latter are artificially changed either during isolation or by methanol fixation. We conclude therefore that parasitic proteins are cryptically arranged in intact host cell plasma membranes of P. chaubaudi-infected erythrocytes.

Isolation and characterization of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi.

A new procedure has been developed which allows the concomitant isolation of viable parasites and host cell plasma membranes from erythrocytes infected with Plasmodium chabaudi trophozoites. The average final yield of parasites is 56%. Free parasites reveal a well preserved ultrastructure, incorporate [14C]isoleucine for at least 3 h, and synthesize about the same proteins as parasites within erythrocytes as monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-autoradiography. The host cell plasma membranes can be isolated in the form of ghosts with an average yield of 27%. The ghosts possess a structurally intact plasma membrane as revealed by freeze-etch electron microscopy. The ghosts are regularly associated with seven neo-proteins as identified by SDS-PAGE and isoelectric focusing (IEF)/SDS-PAGE. These neo-proteins have the following apparent molecular masses: 154 kDa, 145 kDa, 90 kDa, 72 kDa (pI 4.5), 67 kDa, 52 kDa, and 33 kDa (pI 5.7), respectively. The contamination of ghosts by parasite material and, conversely, the contamination of parasites by host cell plasma membranes is very low as demonstrated by light and electron microscopy, lactoperoxidase-mediated radioiodination and the distribution of the typical parasite marker enzymes such as choline kinase, cholinephosphotransferase and ethanolaminephosphotransferase.

Fractionation of Plasmodium chabaudi-infected erythrocytes into parasites and ghosts.

We have developed a new procedure for subfractionation of Plasmodium chabaudi-infected erythrocytes into parasites and ghosts. Trophozoite-infected erythrocytes enriched over a percoll-step (:1.10 g/cm3) are subjected to a glycerol-enhanced osmotic shock. This induces the release of parasites and the emergence of erythrocyte ghosts, which can be separated on a continuous percoll gradient (:1.02----1.10 g/cm3). The parasites are intact in terms of ultrastructure and incorporation of 14C-isoleucine. The erythrocyte ghosts are purified over a two-step percoll gradient (:1.01-1.02 g/cm3). Our method recovers about 40%-50% of the initial ghosts and 70%-95% of the initially freed parasites.