Heat stress-associated changes in the intestinal barrier, inflammatory signals, and microbiome communities in dairy calves.

Recent studies indicate that heat stress pathophysiology is associated with intestinal barrier dysfunction, local and systemic inflammation, and gut dysbiosis. However, inconclusive results and a poor description of tissue-specific changes must be addressed to identify potential intervention targets against heat stress illness in growing calves. Therefore, the objective of this study was to evaluate components of the intestinal barrier, pro- and anti-inflammatory signals, and microbiota community composition in Holstein bull calves exposed to heat stress. Animals (mean age = 12 wk old; mean body weight = 122 kg) penned individually in temperature-controlled rooms were assigned to (1) thermoneutral conditions (constant room temperature at 19.5°C) and restricted offer of feed (TNR, n = 8), or (2) heat stress conditions (cycles of room temperatures ranging from 20 to 37.8°C) along with ad libitum offer of feed (HS, n = 8) for 7 d. Upon treatment completion, sections of the jejunum, ileum, and colon were collected and snap-frozen immediately to evaluate gene and protein expression, cytokine concentrations, and myeloperoxidase activity. Digesta aliquots of the ileum, colon, and rectum were collected to assess bacterial communities. Plasma was harvested on d 2, 5, and 7 to determine cytokine concentrations. Overall, results showed a section-specific effect of HS on intestinal integrity. Jejunal mRNA expression of TJP1 was decreased by 70.9% in HS relative to TNR calves. In agreement, jejunal expression of heat shock transcription factor-1 protein, a known tight junction protein expression regulator, decreased by 48% in HS calves. Jejunal analyses showed that HS decreased concentrations of IL-1α by 36.6% and tended to decrease the concentration of IL-17A. Conversely, HS elicited a 3.5-fold increase in jejunal concentration of anti-inflammatory IL-36 receptor antagonist. Plasma analysis of pro-inflammatory cytokines showed that IL-6 decreased by 51% in HS relative to TNR calves. Heat stress alteration of the large intestine bacterial communities was characterized by increased genus Butyrivibrio_3, a known butyrate-producing organism, and changes in bacteria metabolism of energy and AA. A strong positive correlation between the rectal temperature and pro-inflammatory Eggerthii spp. was detected in HS calves. In conclusion, this work indicates that HS impairs the intestinal barrier function of jejunum. The pro- and anti-inflammatory signal changes may be part of a broader response to restore intestinal homeostasis in jejunum. The changes in large intestine bacterial communities favoring butyrate-producing organisms (e.g., Butyrivibrio spp.) may be part of a successful response to maintain the integrity of the colonic mucosa of HS calves. The alteration of intestinal homeostasis should be the target for heat stress therapies to restore biological functions, and, thus highlights the relevance of this work.

Short communication: Relationships among temperature-humidity index with rectal, udder surface, and vaginal temperatures in lactating dairy cows experiencing heat stress.

The objective of this study was to evaluate relationships between measurements of temperature-humidity index (THI) and rectal, vaginal, and udder surface temperatures in lactating cows exposed to heat stress (HS). In experiment 1, 12 multiparous and 8 primiparous Holstein cows experienced a THI ranging from 69 to 76 at 2000 to 1000 h and THI from 74 to 82 at 1000 to 2000 h (peaked at 82 from 1400 to 1800 h). Cows were exposed to HS 10 h daily for 21 d. Measurements of rectal temperature (RT) and udder surface temperature were collected at 1000 and 1500 h (±30 min). Vaginal temperature was monitored every 10 min using digital loggers, averaged over 1 h, and paired with corresponding rectal and udder surface temperature data. In experiment 2, 12 multiparous Holstein cows experienced a THI ranging from 60 to 76 at 2000 to 1000 h and THI from 69 to 83 at 1000 to 2000 h (peaked at 83 from 1600 and 1900 h), eliciting 10 h/d of HS for 7 d. Rectal and udder surface temperatures were analyzed at 0700 and 1500 h (±30 min). Vaginal temperature was recorded and analyzed as indicated in experiment 1. Afternoon THI showed weak correlations with surface temperature (r = 0.19, n = 420 in experiment 1; r = 0.23, n = 84 in experiment 2), weak to moderate correlations with RT (r = 0.34, n = 366 in experiment 1; r = 0.26, n = 84 in experiment 2), and moderate correlations with vaginal temperature (r = 0.34, n = 175 in experiment 1; r = 0.35, n = 40 in experiment 2). Moreover, vaginal temperature increased 0.10 and 0.22°C per unit of THI (R2 = 0.15 in experiment 1; R2 = 0.40 in experiment 2). Afternoon vaginal temperature strongly correlated with RT (r = 0.69, n = 131 in experiment 1; r = 0.63, n = 37 in experiment 2) and explained 57 (experiment 1) and 68% (experiment 2) of variation in RT. Surface temperature showed moderate to strong correlations with RT (r = 0.57, n = 84) and vaginal temperature (r = 0.74, n = 37) in experiment 2. In conclusion, THI showed a weak to moderate relationship with core body temperatures and explained the increase in rectal and vaginal temperatures experienced by HS cows. Compared with rectal temperature, vaginal temperature showed stronger relationships with THI and can be used to determine thermal load. Udder surface temperature showed a moderate to strong relationship with core body temperature, and this relationship may support the use of surface temperature data to manage thermal load in HS cows.

Lowering rumen-degradable and rumen-undegradable protein improved amino acid metabolism and energy utilization in lactating dairy cows exposed to heat stress.

The objective of this study was to evaluate the effects of reducing dietary rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) on protein and energy metabolism in heat-stressed dairy cows. Eighteen primiparous and 30 multiparous mid-lactation Holstein cows were used in a completely randomized design arranged in a 2 × 2 factorial (n = 12/treatment). Cows were randomly assigned to 1 of 4 dietary treatments that included 2 levels of RDP (10 and 8%; D) and 2 levels of RUP (8 and 6%; U) of dry matter for 21 d as (1) 10D:8U, (2) 8D:8U, (3) 10D:6U, and (4) 8D:6U. Diets were isoenergetic and contained 50% forage and 50% concentrate (dry matter basis). Cows were housed in a freestall barn. Three weeks before start of treatments, all animals were fed the 10D:8U diet and received supplemental cooling to prevent heat stress. During the treatment period, cows experienced a daily increment in temperature-humidity index from 74 to 82 for 1000 to 2000 h. Blood samples were collected on d -1 and 21 of the treatment period to determine plasma concentrations of AA, glucose, insulin, fatty acids, and ß-hydroxybutyrate. For primiparous cows, reducing from 10 to 8% RDP decreased insulin concentrations. For multiparous cows, we found significant RDP by RUP interactions for insulin, ß-hydroxybutyrate, fatty acids, total essential AA, and 3-methylhistidine concentrations. Reducing from 10 to 8% RDP decreased insulin concentrations at 6% RUP, but concentrations did not change when reducing RDP at 8% RUP. Reducing from 10 to 8% RDP decreased ß-hydroxybutyrate concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 10 to 8% RDP increased nonesterified fatty acid and total essential AA concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 8 to 6% RUP decreased 3-methylhistidine concentration at 8% RDP, but not at 10% RDP. Reducing from 8 to 6% RUP increased milk protein yield efficiency in primiparous and multiparous cows. These results indicate that reducing RDP and RUP lowers circulating insulin, which was associated with mobilization and utilization of fatty acids. Reduced RDP and RUP increases the use of AA to maintain milk protein synthesis and limit AA catabolism in cows exposed to warm climates.

Short communication: High incubation temperature in bovine mammary epithelial cells reduced the activity of the mTOR signaling pathway.

Hyperthermia alters utilization of AA in protein synthesis and cell-signaling activity in bovine mammary cells. Essential AA and insulin regulate translation of proteins by controlling the activity of mammalian target of rapamycin (mTOR) signaling pathway. The objectives of this study were to evaluate (1) the effects of incubation temperature on the mTOR signaling pathway and transcription of AA transporters in a bovine mammary alveolar cell line (MAC-T) and (2) the combined effects of incubation temperature and insulin on the mTOR signaling pathway in this cell line. Cells were cultured in medium with 10% fetal bovine serum at 37°C and 5% CO2. In experiment 1, cells were subjected to 37°C (control) or 41.5°C (high incubation temperature; HT) for 12 h. In experiment 2, cells were assigned to 1 of 4 treatments as a 2 × 2 factorial arrangement, including 2 cell culture temperatures (control and HT) and absence or presence of 1.0 µg/mL of insulin. Proteins were harvested and separated by gel electrophoresis. In experiment 1, gene expression of AA transporters (SLC1A1, SLC1A5, SLC3A2, SLC7A1, SLC7A5, and SLC36A1) were evaluated, and changes of ≥2 fold were deemed significantly different. In experiments 1 and 2, immunoblotting was used to identify total and site-specific phosphorylated forms of protein kinase B (Akt1; Ser473), p70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Phosphorylated and total forms of Akt1, S6K1, rpS6, and eEF2 were quantified and expressed as the ratio of phosphorylated to total protein. In experiment 1, HT resulted in a ≥2-fold increase expression of SLC1A1 and SLC3A2. High incubation temperature reduced the phosphorylated to total ratio of Akt1 and rpS6 and increased the phosphorylated to total ratio of eEF2. In experiment 2, we found no temperature by insulin interactions on phosphorylation state of the protein factors of interest. High incubation temperature reduced the phosphorylated to total ratio of Akt1. The addition of insulin increased the phosphorylated to total ratio of Akt1, S6K1, and rpS6. In summary, HT reduced the activity of the mTOR signaling pathway and increased the expression of AA transporters. High incubation temperature possibly reduced protein translation by reducing the mTOR signaling pathway activity in an effort to adapt to thermal stress. These results may help explain the direct effect of elevated temperature on AA metabolism and protein translation in heat-stressed animals.

Lowering rumen-degradable protein maintained energy-corrected milk yield and improved nitrogen-use efficiency in multiparous lactating dairy cows exposed to heat stress.

The objective of this study was to examine the effect of reducing rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) proportions on feed intake, milk production, and N-use efficiency in primiparous and multiparous cows exposed to warm climates. Eighteen primiparous and 30 multiparous mid-lactation Holstein cows were used in a completely randomized design with a 2 × 2 factorial arrangement of treatments. Cows were randomly assigned to 1 of 4 dietary treatments formulated to contain 2 proportions of RDP (10 and 8%) and 2 proportions RUP (8 and 6%) of dry matter (DM) indicated as follows: (1) 10% RDP, 8% RUP; (2) 8% RDP, 8% RUP; (3) 10% RDP, 6% RUP; and (4) 8% RDP, 6% RUP. Protein sources were manipulated to obtain desired RDP and RUP proportions. Diets were isoenergetic and contained 50% forage and 50% concentrate (DM basis). Cows were individually fed the 10% RDP, 8% RUP diet 3 wk before treatment allocation. Cows were exposed to the prevailing Tennessee July and August temperature and humidity in a freestall barn with no supplemental cooling. Main effects and their interaction were tested using the Mixed procedure of SAS (least squares means ± standard error of the mean; SAS Institute Inc., Cary, NC). Observed values of nutrient intake and milk production were used to obtain NRC (2001) model predictions. Cows showed signs of heat stress throughout the study. Reducing from 10 to 8% RDP decreased dry matter intake (DMI; 0.9 kg/d) at 8% RUP, but increased DMI (2.6 kg/d) at 6% RUP in primiparous cows. Reducing from 10 to 8% RDP decreased milk yield (10%) at 8% RUP, but increased yield (14%) at 6% RUP. Treatments did not affect yield of energy-corrected milk. For multiparous cows, treatments did not affect DMI. Reducing from 10 to 8% RDP decreased yield of energy-corrected milk (3.4%) at 8% RUP, but increased yield (8.8%) at 6% RUP. Reducing from 10 to 8% RDP and 8 to 6% RUP both increased N-use efficiency for primiparous and multiparous cows. The NRC model underestimated metabolizable protein and RUP supply, and overestimated RUP requirements, resulting in predictive losses of milk yield 1.4 to 5.8 times greater than observed values. In summary, the reduction of RDP and RUP proportions did not affect DMI, whereas the RUP reduction at 10% RDP had a small negative effect on energy-corrected milk yield. However, reduction of RDP and RUP consistently improved N-use efficiency of heat-stressed multiparous cows. The reduction of RDP and RUP proportions reduced DMI and milk yield but did not affect energy-corrected milk yield in primiparous cows, indicating a limited supply of nutrients.

Drivers of avian habitat use and detection of backyard birds in the Pacific Northwest during COVID-19 pandemic lockdowns.

Birds living in developed areas contend with numerous stressors, including human disturbance and light, noise, and air pollution. COVID-19 pandemic lockdowns presented a unique opportunity to disentangle these effects during a period of reduced human activity. We launched a community science project in spring 2020 to explore drivers of site use by and detection of common birds in cities under lockdown in the U.S. Pacific Northwest. Our goals were twofold: (1) consider how intensity of urbanization, canopy cover, and availability of bird feeders and bird baths influenced avian habitat use; and (2) quantify how daily changes in weather, air pollution, and human mobility influenced detection of birds. We analyzed 6,640 surveys from 367 volunteers at 429 monitoring sites using occupancy models for 46 study species. Neither land cover nor canopy cover influenced site use by 50% of study species, suggesting that backyard birds may have used a wider range of habitats during lockdowns. Human mobility affected detection of 76% of study species, suggesting that birds exhibited species-specific behavioral responses to day-to-day changes in human activity beginning shortly after initial lockdown restrictions were implemented. Our study also showcases how existing community science platforms can be leveraged to support local monitoring efforts.

Physiological responses of Holstein calves to heat stress and dietary supplementation with a postbiotic from Aspergillus oryzae.

Increased ambient temperature causes heat stress in mammals, which affects physiological and molecular functions. We have recently reported that the dietary administration of a postbiotic from Aspergillus oryzae (AO) improves tolerance to heat stress in fruit flies and cattle. Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in part by nutritional interventions. The objective of this study was to characterize the phenotypic response of growing calves to heat stress compared to thermoneutral ad libitum fed and thermoneutral feed-restricted counterparts and examining the physiologic alterations associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on intestinal permeability. In this report, we expand previous work by first demonstrating that heat stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use efficiency, water absorption, and the intestinal permeability in heat stress-mediated increase in gut permeability but did not reduce heat stress-mediated rise in markers of systemic inflammation.

A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms.

Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.

Thrombotic markers in metabolic syndrome subjects exposed to diesel exhaust.

Traffic-derived particulate matter (PM) is associated with cardiovascular morbidity and mortality, but the mechanism of this association is unclear. Prothrombotic processes have been linked to PM in epidemiological and animal models, but have not been consistently implicated in controlled human models. Diesel exhaust (DE) is a major contributor to PM. We conducted a controlled human exposure of DE in subjects with metabolic syndrome. The study objective was to evaluate DE exposure effects on prothrombotic markers in a population vulnerable to cardiovascular disease. A randomized, crossover, double-blinded design was used: 16 subjects with metabolic syndrome exposed on 3 different days (> or = 2 wk washout) to DE at 0 (filtered air, FA), 100 microg PM(2.5)/m(3) (DE(100)) and 200 mug PM(2.5)/m(3) (DE(200)). We assessed DE-associated changes in D-dimer, von Willebrand factor (VWF), and plasmin activator inhibitor-1 (PAI-1) at 3, 7, and 22 h after exposure initiation. A DE(200)-attributable decrease (1.17-fold; CI 1.04 to 1.34) in VWF was noted at 7 h. Significant changes did not occur in other primary endpoints. As previously noted with healthy subjects, strong diurnal patterns in PAI-1 were observed. Thus, in a novel study, we were unable to demonstrate a prothrombotic effect of moderate-dose diesel exhaust exposure in a population at risk for cardiovascular disease.

Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element.

T-cell activation pathways are involved in the regulation of human immunodeficiency virus (HIV) expression. Phorbol 12-myristate 13-acetate (PMA) is a potent inducer of T-cell immune functions and has recently been demonstrated to increase viral replication in cell lines infected with HIV. To define sequences required for viral induction by PMA. T-cell lines were transiently transfected with viral long terminal repeat (LTR) sequences directing chloramphenicol acetyltransferase (CAT) gene expression. PMA added to transfected cell cultures 24 h before harvest reproducibly increased both CAT mRNA and enzyme expression 2- to 2-fold. Sequences necessary for basal and PMA-induced levels of CAT expression were determined by deletion and enhancer reconstitution constructs with fragments and oligonucleotides from the original LTR-CAT expression plasmid. PMA-inducible and basal activity required tandem repeats of a core enhancer element (GGGACTTTCC) located in the LTR between -105 and -82 relative to the RNA start site. The enhancerlike sequence could be inserted at a site distant to the CAT gene open reading frame and functioned in a position- and orientation-independent manner. The data thus define a transcriptionally active regulatory-enhancer element critical to the control of HIV gene expression.

Cardiovascular disease and air pollutants: evaluating and improving epidemiological data implicating traffic exposure.

Evidence suggests that traffic-related pollutants play a role in the observed associations between air pollution and adverse cardiovascular health effects. The contribution of traffic to individual exposures is difficult to quantify in traditional epidemiological studies, however, and researchers have employed various approaches in attempt to isolate its effects. Many investigators have employed ambient measurements such as nitrogen oxides, carbon monoxide, or black carbon as surrogates for traffic in studying associations with health outcomes. Source-apportionment techniques also have been used in a few studies to identify associations with the mixture of pollutants from specific origins, including traffic. In other studies, estimates of traffic near a person's home have predicted cardiovascular endpoints, and local traffic levels have modified the effect of regional air pollution. More recently, studies have linked changes in cardiovascular health to time spent in traffic. In this article, we review the epidemiological evidence regarding the impact of traffic-related pollution on cardiovascular diseases and examine the different techniques used to examine this important research question. We conclude with a discussion of the future directions being used in ongoing epidemiological studies to identify the cardiovascular health impacts of traffic.

Jugular-infused methionine, lysine and branched-chain amino acids does not improve milk production in Holstein cows experiencing heat stress.

Poor utilization of amino acids contributes to losses of milk protein yield in dairy cows exposed to heat stress (HS). Our objective was to test the effect of essential amino acids on milk production in lactating dairy cows exposed to short-term HS conditions. To achieve this objective, 12 multiparous, lactating Holstein cows were assigned to two environments (thermoneutral (THN) or HS) from days 1 to 14 in a split-plot type cross-over design. All cows received 0 g/day of essential amino acids from days 1 to 7 (negative control (NC)) followed by an intravenous infusion of l-methionine (12 g/day), l-lysine (21 g/day), l-leucine (35 g/day), l-isoleucine (15 g/day) and l-valine (15 g/day, methionine, lysine and branched-chain amino acids (ML+BCAA)) from days 8 to 14. The basal diet was composed of ryegrass silage and hay, and a concentrate mix. This diet supplied 44 g of methionine, 125 g of lysine, 167 g of leucine, 98 g of isoleucine and 109 g of valine per day to the small intestine of THN cows. Temperature-humidity index was maintained below 66 for the THN environment, whereas the index was maintained above 68, peaking at 76, for 14 continuous h/day for the HS environment. Heat stress conditioning increased the udder temperature from 37.0°C to 39.6°C. Cows that received the ML+BCAA treatment had greater p.m. rectal and vaginal temperatures (0.50°C and 0.40°C, respectively), and respiration rate (8 breaths/min) compared with those on the NC treatment and exposed to a HS environment. However, neither NC nor ML+BCAA affected rectal or vaginal temperatures and respiration rates in the THN environment. Compared with THN, the HS environment reduced dry matter intake (1.48 kg/day), milk yield (2.82 kg/day) and milk protein yield (0.11 kg/day). However, compared with NC, the ML+BCAA treatment increased milk protein percent by 0.07 points. For the THN environment, the ML+BCAA treatment increased concentrations of milk urea nitrogen. For the HS environment, the ML+BCAA treatment decreased plasma concentrations of arginine, ornithine and citrulline; however, differences were not observed for the THN environment. In summary, HS elicited expected changes in production; however, infusions of ML+BCAA failed to increase milk protein yield. Lower dry matter intake and greater heat load in response to ML+BCAA contributed to the lack of response in milk production in HS cows. The ML+BCAA treatment may have reduced the breakdown of muscle protein in heat-stressed cows.

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

Occupational burns in Washington State, 1989-1993.

Occupational burns cause significant morbidity in the United States each year; however, there are few studies that report industries or workplaces where workers are at an increased risk of burn injuries. Washington State's Department of Labor and Industries (L and I) computerized workers' compensation database was used to describe work-related burns over 5 years. From 1989 to 1993, L and I accepted 27,323 claims for occupational burns, 71.4% of them thermal burns and 26.8% chemical burns. The most common sources of injury were cooking oils (14%) and hot water/steam (13%). Workers involved in food preparation or food handling accounted for the highest proportion of injured workers (30%). Industries involved in the smelting, sintering, or refining of ore had the highest rate for thermal burns, with a rate of 15.0 burn injuries per 100 full-time equivalent workers per year, followed by paper, pulp, or wood fiber manufacturing, with a rate of 5.8, then roof work, with a rate of 4.3. Industries involved in hazardous waste landfill clean-up had the highest rate for chemical burns, with a rate of 4.9, followed by portable cleaning and washing, with a rate of 3.5, and paper, pulp, and wood fiber manufacturing, with a rate of 2.6. Further study is needed to identify work practices that result in burn injuries in order to decrease the incidence of this preventable occupational injury.

Washington State's late night retail worker crime protection regulation. Relationships with employer practices.

Washington's late night retail worker crime protection regulation, enforced by the state Occupational Safety and Health Administration (OSHA) program, was intended to prevent injuries by deterring violent crimes. We investigated whether the regulation was associated with businesses' violence prevention activities. We surveyed 1,516 employers at high risk of robbery, including gas stations, groceries, hotels, restaurants, and taverns, in 1995 to determine whether they had violence prevention training programs for their employees (a requirement of the standard). Awareness of the regulation was low (4.4%). Employers covered by the regulation were more likely to have programs (Odds Ratio [OR] = 1.4), as were those aware of a regulation (OR = 3.4). State OSHA plan contact (an inspection or consultation) was also associated with having a program (OR = 1.9). Despite low awareness of the standard, results suggested that regulatory efforts to protect high-risk employees were associated with employers' robbery and crime prevention activities.

Surveillance of occupational diseases in the United States. A survey of activities and determinants of success.

Managers of state-based occupational disease surveillance programs were interviewed for information on their program's characteristics and factors that contributed to their success. There were 68 programs in 52 jurisdictions (50 states, the District of Columbia and New York City). Reportable conditions ranged from a specific disease to "all occupational diseases". Of these programs, 56% met at least one of their objectives. Conditions associated with successful programs usually had short latency periods, were easily diagnosed, and were related to a workplace hazard. They included agricultural injuries, burns, respiratory diseases, cumulative trauma disorders, and poisonings due to lead, pesticides, or carbon monoxide. Successful programs had larger budgets and more staff than did unsuccessful programs, and also took actions after notification of a condition.

Health symptoms and the work environment in four nonproblem United States office buildings.

OBJECTIVES: The objective of this study was to quantify health symptom reports in four "nonproblem" buildings and to assess the relationship between symptoms and air quality measures, workstation characteristics, and psychosocial aspects of the workplace. METHODS: Environmental sampling was conducted in four office buildings occupied by employees working for the state of Washington in March of 1992. A questionnaire was concurrently administered to building occupants. RESULTS: While measured contaminant levels were low, symptoms were frequent. Fifty-five percent of the 646 respondents reported recent symptoms which affected the eyes, nose, or throat and improved when away from work. Symptoms were not associated with measured contaminant levels, but, rather, with perceptions about air movement, dryness, odors, and noise. Psychosocial factors were less strongly associated with symptoms. CONCLUSIONS: Even in nonproblem buildings, symptom reports can be frequent and may represent overall satisfaction with the work environment. In response to symptoms ascribed to air quality problems, it may be appropriate to address employee perceptions regarding the work environment in addition to evaluating environmental characteristics relating to chemicals, biological contamination, air movement, temperature, and humidity.

Latex sensitivity in Washington State acute care hospitals. A needs assessment and survey of awareness of the issues.

Based on reports from the workers' compensation system and a sentinel health provider network, latex gloves may be one of the most prevalent sources of occupational skin disorders in Washington State's health care industry. To gather information to understand and address this problem, questionnaires were distributed to 105 acute care hospitals in Washington State. Employee health and infection control specialists were queried on their knowledge about latex allergy, the perceived extent of the problem, and the actions taken to address the problem. With 95 of the hospitals returning completed questionnaires (93% response rate), 30% reported having problems with latex allergies among employees in their facility, with most reporting two or fewer cases. Adequate knowledge was found about the causes and effects of latex allergies, but definite knowledge gaps existed. More than 60% of all of the hospitals surveyed had made some type of glove alternatives available to affected employees, 4% had designated latex free zones, 4% had cleaned to remove latex dust, and 7% had done nothing to address the issue.