EARLY RESPONSE OF RAT BRAIN TRYPTOPHAN HYDROXYLASE ACTIVITY TO CYCLOHEXIMIDE, PUROMYCIN AND CORTICOSTERONE

Abstract— The activity of tryptophan hydroxylase was measured in whole homogenates of midbrain and forebrain areas of the rat brain. A significant elevation of tryptophan hydroxylase in midbrain and forebrain was found within 1 h after injection of corticosterone hemisuccinate Na salt (10mg/kg) into normal rats. A further elevation of tryptophan hydroxylase at 4 h after injection occurred only in the midbrain region. A rapid alteration of tryptophan hydroxylase was also observed following intracistemal injection of a protein synthesis inhibitor, cydoheximide. A significant depression of 50% of normal levels occurred both in midbrain and forebrain regions within 1 h. However. 4 h after injection only the midbrain tryptophan hydroxylase level was depressed, and this depression was 16% of normal levels. This temporal and spatial pattern following cydoheximide injection was not the result of changes in the ability of cydoheximide to inhibit in vivo protein synthesis since [³H]valine incorporation into protein was shown to be equally depressed at both 1 and 5 h in both the midbrain and forebrain. Puromycin blocked [³H]valine incorporation into proteins in the midbrain and forebrain. but only caused a depression of 16% of tryptophan hydroxylase in the midbrain at 4 h. The aminonucleoside derivative of puromycin has no effect on protein synthesis or on tryptophan hydroxylase. Cydoheximide had no effect on tryptophan hydroxylase in vitro. The data suggest that cydoheximide and corticosterone produce an early (1 h) effect on tryptophan hydroxylase unrelated to de novo protein synthesis in regions known to contain perikaryon (midbrain) and axon terminals (forebrain) of 5-HT-containing neurons. The later (4h) effects of these two compounds and puromycin on tryptophan hydroxylase in the perikaryon (midbrain) region of 5-HT-containing neurons probably result from alteration in de novo protein synthesis. The half time of tryptophan hydroxylase in midbrain region is calculated to be 12 h.     

QUANTITATIVE ASSESSMENT OF LEFT VENTRICULAR ASYNERGY

Myocardial performance in the intact human heart can be assessed from the analysis of ejection phase indices. Accordingly, among 20 consecutive patients who were studied by means of biplane left ventricular cineangio-cardiography, 18 were selected solely on the basis of high quality angiograms. The characteristics of left ventricular contraction were expressed quantitatively by the systolic ejection fraction, the mean velocity of circumferential fiber shortening at the left ventriculalr equator, and at several chords, the mean velocity of shortening of the hemichords and the mean normalized systolic ejection rate. All 18 patients had abnormalities of contraction based on the velocity of the hemichords. Both ejection fraction and mean normalized systolic ejection rate showed a low sensitivity in detecting depressed myocardial function in patients with segmental asynergy. Equatorial V(CF) provided additional information only when the affected areas were adjacent to the left ventricular minor axis. The sensitivity of this index was markedly increased by construction of several chords perpendicular to the left ventricular long axis (segmental V(CF)). However, when only one wall was affected, measurement of the velocity of shortening of the hemichords provided a better definition of the regional performance.     

Fire effects and ecological recovery pathways of tropical montane cloud forests along a time chronosequence

Tropical montane cloud forests (TMCFs) harbour high levels of biodiversity and large carbon stocks. Their location at high elevations make them especially sensitive to climate change, because a warming climate is enhancing upslope species migration, but human disturbance (especially fire) may in many cases be pushing the treeline downslope. TMCFs are increasingly being affected by fire, and the long‐term effects of fire are still unknown. Here, we present a 28‐year chronosequence to assess the effects of fire and recovery pathways of burned TMCFs, with a detailed analysis of carbon stocks, forest structure and diversity. We assessed rates of change of carbon (C) stock pools, forest structure and tree‐size distribution pathways and tested several hypotheses regarding metabolic scaling theory (MST), C recovery and biodiversity. We found four different C stock recovery pathways depending on the selected C pool and time since last fire, with a recovery of total C stocks but not of aboveground C stocks. In terms of forest structure, there was an increase in the number of small stems in the burned forests up to 5–9 years after fire because of regeneration patterns, but no differences on larger trees between burned and unburned plots in the long term. In support of MST, after fire, forest structure appears to approximate steady‐state size distribution in less than 30 years. However, our results also provide new evidence that the species recovery of TMCF after fire is idiosyncratic and follows multiple pathways. While fire increased species richness, it also enhanced species dissimilarity with geographical distance. This is the first study to report a long‐term chronosequence of recovery pathways to fire suggesting faster recovery rates than previously reported, but at the expense of biodiversity and aboveground C stocks.     

EXERCISE TESTING AND LEFT MAIN CORONARY ARTERY DISEASE: EXPERIENCE WITH 57 PATIENTS

Results of multistage treadmill tests (TMT) of 57 patients with critical stenosis (>/= 50%) of the left main coronary artery were analyzed. Additional disease was present in the major vessel in three patients (5%), two vessels in 18 patients (32%), and three vessels in 35 patients (61%). The TMT was negative for ischemia in only two patients (4%), positive in 51 (89%), and undetermined in 4 (7%). TMT was strongly positive (>/= 2 mm ST segment depression) in 40 patients (70%), and in 11 (19%) of these, ST depression was >/= 3 mm. Hypotension with exercise was rare and was encountered in only one patient. Arrhythmias were induced with exercise in six patients (10%) and resulted in premature termination of TMT in four. TMT was terminated due to early ST segment depression in 40 patients (70%), in 17 (30%) without chest pain-an unusual finding. Exercise was limited to stage I (Bruce protocol) in 16 (28%), stage II in 26 (46%), stage III in ten (17%), and stage IV in five (9%). Mean exercise tolerance was 298 +/- 22 seconds (SEM). Maximum heart rate (HR) achieved was 76 +/- 2% of their maximal predicted values. Peak double product (systolic BP x HR) was 20490 +/- 830. The data suggest that the TMT is rarely negative in the presence of LM lesions. An early strongly positive response with or without pain should lead one to suspect LM disease. Exercise-induced hypotension is rare. Limited exercise tolerance and/or early ST segment depression in stages I and II of TMT seem to be predictive of the severity of LM lesions.     

SEVERE PULMONARY HYPERTENSION WITH SIGNIFICANT "A" DIP ON PULMONIC VALVE ECHOCARDIOGRAM

A patient with severe pulmonary hypertension and no evidence of right ventricular failure who had a 4 mm "a" dip on the pulmonic valve echocardiogram is reported. Although other echocardiographic abnormalities suggesting pulmonary hypertension were recorded in our patient, the normal "a" dip of the pulmonic valve in the absence of right ventricular failure appears to be an exception to previously reported findings. We suggest motion of the entire pulmonary artery as an explanation for this phenomenon.     

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.     

Arboviral Etiologies of Acute Febrile Illnesses in Western South America, 2000–2007

Background

Arthropod-borne viruses (arboviruses) are among the most common agents of human febrile illness worldwide and the most important emerging pathogens, causing multiple notable epidemics of human disease over recent decades. Despite the public health relevance, little is know about the geographic distribution, relative impact, and risk factors for arbovirus infection in many regions of the world. Our objectives were to describe the arboviruses associated with acute undifferentiated febrile illness in participating clinics in four countries in South America and to provide detailed epidemiological analysis of arbovirus infection in Iquitos, Peru, where more extensive monitoring was conducted.

Methodology/Findings

A clinic-based syndromic surveillance system was implemented in 13 locations in Ecuador, Peru, Bolivia, and Paraguay. Serum samples and demographic information were collected from febrile participants reporting to local health clinics or hospitals. Acute-phase sera were tested for viral infection by immunofluorescence assay or RT-PCR, while acute- and convalescent-phase sera were tested for pathogen-specific IgM by ELISA. Between May 2000 and December 2007, 20,880 participants were included in the study, with evidence for recent arbovirus infection detected for 6,793 (32.5%). Dengue viruses (Flavivirus) were the most common arbovirus infections, totaling 26.0% of febrile episodes, with DENV-3 as the most common serotype. Alphavirus (Venezuelan equine encephalitis virus [VEEV] and Mayaro virus [MAYV]) and Orthobunyavirus (Oropouche virus [OROV], Group C viruses, and Guaroa virus) infections were both observed in approximately 3% of febrile episodes. In Iquitos, risk factors for VEEV and MAYV infection included being male and reporting to a rural (vs urban) clinic. In contrast, OROV infection was similar between sexes and type of clinic.

Conclusions/Significance

Our data provide a better understanding of the geographic range of arboviruses in South America and highlight the diversity of pathogens in circulation. These arboviruses are currently significant causes of human illness in endemic regions but also have potential for further expansion. Our data provide a basis for analyzing changes in their ecology and epidemiology.     

Induction of a Cross-Reactive CD8+ T Cell Response following Foot-and-Mouth Disease Virus Vaccination

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8⁺ T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8⁺ T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8⁺ T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8⁺ T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αβ CD8⁺ T cells, but not CD4⁺ T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8⁺ T cells from FMDV serotype O-vaccinated A31⁺ cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8⁺ T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes.Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae, genus Aphthovirus. The FMDV particle consists of a positive-strand RNA molecule of approximately 8,500 nucleotides, enclosed within an icosahedral capsid. The genome encodes a unique polyprotein from which four structural proteins (P1A, P1B, P1C, and P1D; also referred to as VP4, VP2, VP3, and VP1, respectively) and nine nonstructural proteins are cleaved by viral proteases (48). FMDV shows a high genetic and antigenic variability, which is reflected in the seven serotypes and multiple subtypes reported so far (13). The virus causes a highly contagious infection in cloven-hoofed animals which is characterized by the formation of vesicles on the mouth, tongue, nose, and feet. In addition, most infected animals develop viremia.The virus elicits a rapid humoral response in both infected and vaccinated animals (26). Virus-specific antibodies protect animals in a serotype-specific manner against reinfection or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies (38). Control of the disease is achieved by vaccination with a chemically inactivated whole-virus vaccine emulsified with adjuvant; however, this provides only short-term, serotype-specific protection (2). The introduction of this vaccine has been very successful in areas of the world where the disease is enzootic. However, one of the major difficulties in implementing vaccination is the inability to distinguish vaccinated animals from infected/recovered animals, which may still be shedding virus. Currently, a number of assays specifically developed for this purpose are being validated (29, 41), and the success of these assays is dependent on the use of purified vaccine antigen. A strategy using replication-deficient adenovirus 5 expressing FMDV antigens has been shown to provide early protection against homologous challenge (39).The identification and characterization of T cell epitopes are important for understanding protective immunity mediated by CD8⁺ and CD4⁺ T lymphocytes. Such T cell responses are pathogen specific and are restricted by major histocompatibility complex (MHC) class I and class II molecules, which present foreign peptides to the immune system (55, 56). The role of cellular immunity in the protection of animals from FMDV is still a matter of some controversy. Specific T cell-mediated antiviral responses have been observed in cattle and swine following either infection or vaccination (3, 7, 24). CD4⁺ T cell responses are suggested to play an important role in protection against FMDV, and published studies demonstrate the presence of FMDV-specific MHC class II-restricted responses in cattle and pigs (22, 24). CD4⁺ epitopes within both P1A and P1D proteins have recently been identified in cattle (23). We have recently reported the presence of FMDV-specific, MHC class I-restricted CD8⁺ T cell responses in cattle following infection or vaccination. Despite these observations, the significance of cell-mediated immune responses in protective immunity to FMDV remains unclear.Cattle MHC (bovine leukocyte antigen [BoLA]) is relatively complex, with variable haplotypes expressing one, two, or three of the six classical class I genes (6, 15). At present, about 60 full-length validated cattle MHC class I cDNA sequences have been identified (www.ebi.ac.uk/ipd/mhc/bola), and the haplotypes commonly found in the Holstein breed are well characterized. We have previously identified amino acid motifs present in peptides binding to BoLA class I alleles N*02101, N*02201, and N*01301 (20). More recently, a number of Theileria parva CD8⁺ T cell epitopes presented through these and additional class I alleles have been described (25). Identification of such epitopes allows detailed analysis of cellular immune responses to vaccination and infection.In the present study, we aimed to identify MHC class I-restricted CD8⁺ T cell epitopes within the FMDV capsid protein. Using a panel of overlapping peptides, we have identified a BoLA A31-restricted epitope that is similar in all FMDV serotypes.