fos-1, a putative histidine kinase as a virulence factor for systemic aspergillosis.

In fungi, two-component histidine kinases have various functions including regulation of osmosensitivity, and of cell-wall assembly. Furthermore, one of these proteins, cos-1, has been shown to be important for virulence of Candida albicans. Recently, a putative histidine kinase, fos-1, has been isolated and partially characterized from Aspergillus fumigatus. Here we compare the virulence of a fos-1 deletion strain with that of the parental wild-type strain in a murine model of systemic aspergillosis. Our results show that the fos-1 deletion strain has significantly reduced virulence as compared with the parental wild-type strain. Thus, we propose that the fos-1 two-component histidine kinase is a virulence factor of A. fumigatus.

COS-l, a putative two-component histidine kinase of Candida albicans, is an in vivo virulence factor.

The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.

The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus.

In fungi, two-component histidine kinases are involved in response mechanisms to extracellular changes in osmolarity, resistance to dicarboximide fungicides, and cell-wall assembly. In the human opportunistic fungus, Candida albicans, each of the three histidine kinases plays a role in virulence. Here, we identify, for the first time, a gene, FOS-1, from the human pathogenic fungus Aspergillus fumigatus that predicts a protein with homology to two-component histidine kinases. The predicted FOS-1 protein is highly homologous to bacterial and other fungal histidine kinases in several functional domains, but is divergent at the amino- and carboxy-termini. A mutant lacking the FOS-1 locus, DeltaFOS-1, did not exhibit a detectable defect in either hyphal growth or morphology when grown on solid or liquid medium. However, in liquid medium, conidiophore development of the DeltaFOS-1 mutant was delayed. Compared to wild type, the DeltaFOS-1 strain was neither osmotically sensitive nor sensitive or resistant to a number of nondicarboximide antifungal drugs, but was highly resistant to dicarboximide fungicides and resistant to novozym 234, suggesting that FOS-1p may play a role in the regulation of cell-wall assembly.

Characterization and optimization of in vitro assay conditions for (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening.

(1,3)Beta-D-glucan synthase (E.C.2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyl transferase) catalyzes the polymerization of glucose ([1-3]-beta-linkages) using UDP-glucose as substrate. We have determined optimal in vitro conditions for the assay of (1,3)beta-glucan synthase activity from Aspergillus fumigatus and Candida albicans. These included lysis of cells in the following for C. albicans, 100 mM HEPES, pH 8.0, 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), 2 mM ethylenediaminetetraacetic acid (EDTA), disodium salt, 5 mM NaF, 250 mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50 mM HEPES, 10mM EDTA, 750 mM sucrose, 10 mM NaH2PO4, 100 mM cellobiose and 50 microM GTPgammaS. Resulting low-speed supernatants were used as enzyme sources to determine the optimal in vitro assay conditions. We have characterized the resulting enzyme activities and tested the optimized assays with known (1,3)beta-glucan synthase inhibitors including cilofungin, papulacandin, aculeacin A, and echinocandin B. We have used both optimized assays to screen > 1000 extracts of marine macroorganisms and, using bioassay-guided purification, have identified (1,3)beta-glucan synthase inhibitors.

Inhibition of Neurospora crassa growth by a glucan synthase-1 antisense construct.

We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-glucan synthase [E.C. 2.4.1.34; UDPglucose: 1,3-beta-D-glucan 3-beta-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)beta-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)beta-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.

Effect of form of nitrogen on growth of ruminal microbes in continuous culture.

A study was conducted to determine the effect of various forms of N on the growth of ruminal microbes in a continuous culture system with solids and liquid dilution rates comparable to those of a high-producing dairy cow. Nitrogen forms were isolated soy protein, soy peptides, individual amino acids (AA) blended to profile soy protein, and urea, which were fed alone and in combinations so that the total N provided was 1.6% of the diet DM. The 100% soy protein treatment resulted in reduced digestion of N and nonstructural carbohydrate compared with other N forms, and outflow of bacterial N/24 h was less than when peptides were fed. This suggested that proteolysis rather than peptide uptake was the rate-limiting step in N utilization in this study. Non-urea N forms increased ADF digestion, total VFA production and the molar percentages of isobutyrate, isovalerate, and valerate compared to urea, which reflected the contribution of carbon skeletons of AA. When combinations of N forms were used, each form contributed an equal quantity of N, 50% of the total treatment, which was .8% of the diet DM. Combinations of N forms did not enhance, and in most cases reduced, ADF and NDF digestion when compared with individual N forms, and no combinations increased microbial growth over that of the individual forms. These results confirm that N forms other than ammonia are needed not only for maximum microbial growth, and they further demonstrate a need for non-protein N for the fiber digestion. In addition, results of this study suggest a requirement for a minimum level of peptide or AA N, which was met only when individual N forms were fed.

Tibial eminence fracture with meniscal entrapment.

Controversy has existed regarding the treatment of tibial eminence fractures in children. It has been widely assumed that closed manipulation of the knee resulting in radiographic reduction of the fragment is acceptable. We present a case report of a trapped medial meniscus, even after apparent successful closed reduction of an incompletely displaced tibial eminence fracture. The integrity of meniscii cannot be inferred radiographically. Arthroscopic evaluation allows reduction of a trapped meniscus and suture fixation of the fragment is easily accomplished.

Inhibition of the in vitro development of Eimeria tenella in chick kidney cells by immune chicken splenocytes.

An in vitro microbicidal assay was used to study the immune response of chickens to Eimeria tenella by measuring the effect of splenocytes from immunized chickens on intracellular development of E. tenella. Splenocytes were prepared from specific-pathogen-free chickens [strain P2a(B19B19) or N2a(B21B21)], immunized one, two, or three times with non-lethal doses of E. tenella. Twelve hours following infection of chick kidney cells (CKCs) with E. tenella sporozoites, splenocytes were added to infected CKCs for 4, 8, or 36 hours. Intracellular E. tenella development was allowed to continue until 72 hours after sporozoite infection, when intracellular development was quantitated by counting merzoites. Immune splenocytes significantly inhibited E. tenella intracellular development after one, two, or three immunizations. Significant inhibition occurred with 4, 8, or 36 hours of coculture and was no greater with longer co-culture times. Immune P2a splenocytes significantly reduced merozoite development in both syngeneic P2a and allogeneic N2a infected CKCs, whereas immune N2a splenocytes had little effect on E. tenella development in either N2a or P2a infected CKCs. These results suggest that immune splenocytes are induced and act relatively rapidly and are not apparently restricted by the major histocompatibility complex, consistent with natural killer cell activity.

Auditory perception, phonological processing, and reading ability/disability.

Auditory perception has been proposed as one source of individual variation in the phonological abilities that play a critical role in skilled reading as well as in reading disabilities. A structural equation approach (LISREL, Jöreskog & Sörbom, 1990) was used to analyze relationships among auditory perception, phonological processing, and reading in a sample of 94 college undergraduates, 24 of whom met specific criteria for a reading disability. In the mathematical model that proved to be the best fit to the data, speech perception was strongly related to three of four phonological variables including short- and long-term auditory memory and phoneme segmentation. These phonological variables in turn were strongly related to reading. Nonverbal temporal processing was not significantly related to any of the phonological variables in the structural equations. It was concluded that speech perception, which was measured with speech repetition, syllable sequence discrimination, and degraded speech tasks, may contribute significantly to individual differences in the phonological abilities necessary for skilled reading.

Interactions of fiber and nonstructural carbohydrates on lactation and ruminal function.

Four Holstein cows averaging 147 DIM and fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square trial to determine diet effects on DMI, milk production, and ruminal metabolism. Diets contained either rapidly or slowly degraded NDF, referred to as low fill and high fill, respectively, combined with two percentages of nonstructural carbohydrate. Treatments were 39% nonstructural carbohydrate (low or high fill) and 29% nonstructural carbohydrate (low or high fill). Intake of DM was not affected by either fill or nonstructural carbohydrate. Ruminal NDF digestibilities averaged 43.1 and 35.6% for the low fill and high fill diets, respectively. Ruminally digested nonstructural and total carbohydrate increased, but milk production decreased, as nonstructural carbohydrate increased from 29 to 39% in diets. Liquid and solid ruminal passage rates, as measured by Co-EDTA and Yb, respectively, were reduced by either 39% nonstructural carbohydrate or low fill diets. Lower microbial N flow to the duodenum and lower efficiency of microbial growth also were observed for diets with 39% nonstructural carbohydrate. The combination of 39% nonstructural carbohydrate and rapidly degraded fiber gave the highest DM and nonstructural carbohydrate digestion in the rumen but resulted in low microbial N synthesis per day and the least microbial N per kilogram of OM digested.

Neospora caninum infection in English Springer Spaniel littermates. Diagnostic evaluation and organism isolation.

Progressive paraparesis developed in four male English Springer Spaniel pups from a litter of five during the first 10 weeks of life. Two of the pups, which had the earliest onset of neurologic signs, were euthanatized without further workup. However, a detailed investigation was completed on the remaining two littermates at 12 weeks of age. Both pups had progressive paraparesis for 3 to 4 weeks before presentation, with one dog developing subsequent asymmetric pelvic limb extensor rigidity. Based on results from neurologic examination, cerebrospinal fluid (CSF) analysis, electrophysiology, and muscle/nerve biopsy, a presumptive diagnosis of protozoal polyradiculitis and polymyositis was made. Necropsy of the most severely affected pup confirmed the clinical diagnosis of inflammatory nerve root and muscle disease but no organisms were found. To increase the potential yield of organisms, the second pup was placed on immunosuppressive doses of corticosteroids and euthanatized 2 weeks later. Numerous organisms were found in lesions in muscle and the central nervous system. Organisms grew in tissue culture and were isolated from the peritoneal fluid of gerbils inoculated with infected tissue. Organisms were not isolated from inoculated mice, guinea pigs, rabbits, and hamsters. No parasites were seen in feces or tissues of three cats fed infected dog tissues. Serologic testing demonstrated a strong positive titer to Neospora caninum in both pups, and electron microscopy showed the characteristic morphology of this parasite.

Rumen digestive physiology and microbial ecology.

The rumen is a dynamic, continuous fermentation compartment that provides a suitable environment for a variety of species of anaerobic bacteria, protozoa, and fungi. These microorganisms have a complex series of interactions with the feeds supplied to the host, with some using particulate matter as both sources of nutrients and sites of sequestration to avoid being washed from the rumen by the rapid flow of fluids. Because of the ability to use soluble nutrients and to reproduce rapidly, other microbes associate primarily with the liquid phase of the rumen contents. Due to the metabolic activity of all microbial populations, feeds are converted to microbial matter and fermentation end products, which serve as nutrients for the ruminant. Optimum feed utilization by ruminants is dependent on achieving maximum rumen fermentation and flow of microbial protein to the duodenum. At this time, it is clear that the major nutrients required by the microbial populations include both fibrous and nonfibrous sources of carbohydrates and nitrogen in the form of ammonia, amino acids, and peptides. In spite of five decades of research, the exact quantities and sources of these nutrients that will result in optimum rumen fermentation rates and microbial yields are only partially known.

Impact of carbohydrate and protein levels on bacterial metabolism in continuous culture.

Diets formulated with three levels of nonstructural carbohydrate (54, 37, and 25% of DM), with various concentrations of degradable intake protein ranging from 19 to 4% of DM, were fermented in continuous cultures to ascertain the effects of ratio of nonstructural carbohydrate to degradable intake protein on bacterial metabolism. Fermenters were maintained at a dilution rate of 12%/h with a solids retention time of 24 h. Regardless of degradable intake protein level, bacterial efficiency (g of bacterial N/kg of DM digested) and VFA production (mM/d) were lower for diets with 25% nonstructural carbohydrate compared with the 37 and 54% nonstructural carbohydrate diets. In response to widening nonstructural carbohydrate:degradable intake protein ratios, bacterial efficiencies at all nonstructural carbohydrate levels declined quadratically from 34.2 to 10.3 with the lowest efficiencies on the 25% nonstructural carbohydrate diets. Bacterial protein production, DM digestion, NDF digestion, and VFA production (mM/d) increased linearly in response to dietary protein. The enhanced NDF and DM digestion, VFA production, and bacterial efficiencies observed with the narrower ratios of nonstructural carbohydrate:degradable intake protein support the theory that level of both degradable intake protein and nonstructural carbohydrate should be considered in order to enhance ruminal digestion and bacterial N production.

Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein.

Three ruminally and duodenally cannulated, lactating Holstein cows were used in a 3 x 3 Latin square experiment to study the effects of differing levels of nonstructural carbohydrate and degradable intake protein on ruminal digestibility and microbial protein production. Three diets were formulated to contain 1) 38 and 13.2%, 2) 31 and 11.8%, and 3) 24 and 9% nonstructural carbohydrate and degradable intake protein as percentages of the DM, respectively. Dry matter intakes were similar for all diets (21.9, 21.1, and 18.3 kg/d for diets 1, 2, and 3, respectively). Likewise, microbial efficiency, as estimated from purine analysis, was unaffected by diet and averaged 24 g of microbial N/kg of OM digested for all treatments. Ruminal digestion of OM averaged 66.6, 65.1, and 55.7% for diets 1, 2, and 3, respectively, resulting in lower microbial N flow per day for diet 3 (317, 333, and 202 g, respectively). Digestion of nonstructural carbohydrate and CP followed similar trends as did OM digestion, whereas NDF digestion remained similar across all diets. These results indicate that nonstructural carbohydrate greater than 24% and ruminally degradable protein greater than 9% of DM will enhance microbial protein flow from the rumen.

Effects of fish meal protein supplementation on milk yield and composition and blood constituents of dairy cows.

Ten holstein and 10 Ayrshire cows were fed diets containing undegradable intake protein from either fish meal or corn gluten meal. Cows were introduced to diets 10 d before projected calving date and individually fed blended rations until 60 d postpartum. Diets were balanced for NE1, CP, and degradable and undegradable protein. Source of undegradable protein did not affect total or FCM yields, DM intake, or milk protein percentage. Cows on fish meal diets tended to lose less BW than those on the corn gluten meal supplement (5.3 vs. 10.3% loss of initial BW). Fish meal supplementation resulted in decreased milk fat and SNF percentage (3.2 vs. 4.2% and 8.37 vs. 8.65%, respectively), but diet did not affect total milk fat, protein, or SNF yield. Plasma nonesterified fatty acids decreased and serum insulin increased with increasing weeks postpartum but were not affected by diet. In this study, no significant advantage was found to using fish meal as a source of undegradable intake protein and feed cost was higher when it was used.

Graph partitioning using annealed neural networks.

A new algorithm, mean field annealing (MFA), is applied to the graph-partitioning problem. The MFA algorithm combines characteristics of the simulated-annealing algorithm and the Hopfield neural network. MFA exhibits the rapid convergence of the neural network while preserving the solution quality afforded by simulated annealing (SA). The rate of convergence of MFA on graph bipartitioning problems is 10-100 times that of SA, with nearly equal quality of solutions. A new modification to mean-field annealing is also presented which supports partitioning graphs into three or more bins, a problem which has previously shown resistance to solution by neural networks. The temperature-behavior of MFA during graph partitioning is analyzed approximately and shown to possess a critical temperature at which most of the optimization occurs. This temperature is analogous to the gain of the neurons in a neural network and can be used to tune such networks for better performance. The value of the repulsion penalty needed to force MFA (or a neural network) to divide a graph into equal-sized pieces is also estimated.

Effects of fish meals on rumen bacterial fermentation in continuous culture.

Effects of various forms of fish meal on microbial metabolism were investigated in continuous cultures of rumen contents. Five diets were formulated to contain 12% ruminally degradable protein and 47 to 48% nonstructural carbohydrate. Soybean meal was the major protein source in the control diet, whereas in the other four diets, various fish meals were substituted for 6% of total diet DM. Fish meals were: fish meal containing 34.4% FFA, fish meal containing 34.4% FFA with CaCl2 added, fish meal containing 65.6% FFA, and fish meal defatted using 1:1 ethanol:ether extraction. The five treatments were fermented with pH either held constant at 6.2 or not controlled. When pH was maintained at 6.2, the inclusion of any fish meal except defatted fish meal reduced the acetate:propionate ratio, decreased protein digestion, and reduced microbial N produced/per kilogram DM digested when compared with the soybean control. When not controlled, pH decreased after feeding to 6.0 or lower. Under these conditions, the soybean control had a lower acetate:propionate ratio and lower NDF digestion than all diets containing fish meal. In this study, oil-containing fish meal affected microbial metabolism more negatively when the fermentation pH was held at 6.2 than when the pH was 6.0 or lower.

Cardiovascular effects of acupuncture stimulation at point Governing Vessel 26 in halothane-anesthetized ponies.

The acupuncture point Governing Vessel (GV-26) is used commonly in the Orient to treat shock in human beings and other species. The cardiovascular effects of stimulation of GV-26 were studied in healthy ponies during 2 episodes of halothane anesthesia. During one anesthetic episode, electrical stimulation (electrostimulation) of GV-26 was performed, and during the other anesthetic episode, heat stimulation (moxibustion) of GV-26 was performed. The order of the stimulations was random. A nonacupuncture point was selected for comparable control stimulation during each experiment. Control and acupuncture stimulations were of equal intensity. There were no significant changes in cardiac output, systemic arterial pressure, or heart rate associated with either electrical or heat acupuncture stimulation.

A microprocessor-based instrument to measure pulmonary functions in critically-ill newborn infants.

Despite advances in the medical care of critically-ill newborn infants, chronic complications due to intense ventilatory support are common. Selection of ventilator settings is empirical, since measurement of pulmonary functions in newborn infants is not commonly performed. A microprocessor-based instrument was constructed to measure pulmonary function in critically-ill newborn infants with respiratory failure. Data collected fail to demonstrate any beneficial effect upon pulmonary mechanics with increasing amounts of continuous distending airway pressure, and in fact demonstrate an adverse effect upon the lung ventilation. These results demonstrate the need to measure pulmonary function to develop procedures to select optimum ventilator settings. A microprocessor-based system is suitable for such an application, and could be incorporated into future infant respiratory support equipment.