Effects of grain, fructose, and histidine on ruminal pH and fermentation products during an induced subacute acidosis protocol.

The effects of grain, fructose, and histidine on ruminal pH and fermentation products were studied in dairy cattle during an induced subacute acidosis protocol. Thirty Holstein heifers were randomly allocated to 5 treatment groups: (1) control (no grain); (2) grain [fed at a crushed triticale dry matter intake (DMI) of 1.2% of body weight (BW)]; (3) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI); (4) grain (1.2% of BW DMI)+histidine (6 g/head); and (5) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI)+histidine (6 g/head) in a partial factorial arrangement. Heifers were fed 1 kg of grain daily with ad libitum access to ryegrass silage and alfalfa hay for 10 d. Feed was withheld for 14 h before challenge day, on which heifers were fed 200 g of alfalfa hay and then the treatment diets immediately thereafter. Rumen samples were collected 5 min after diet ingestion, 60 min later, and at 3 subsequent 50-min intervals. Grain decreased ruminal pH and increased ammonia, total volatile fatty acid (VFA), acetate, butyrate, propionate, and valerate concentrations compared with controls. The addition of grain had no effect on ruminal D- and L-lactate concentrations. Fructose markedly decreased ruminal pH and markedly increased D- and L-lactate concentrations. Fructose increased total VFA and butyrate and decreased valerate concentrations. Although histidine did not have a marked effect on ruminal fermentation, increased concentrations of histamine were observed following feeding. This study demonstrates that the substitution of some grain for fructose can lower ruminal pH and increase VFA and lactate concentrations, warranting further investigation into the role of sugars on the risk of acidosis in dairy cattle.

Adiposity and plane of nutrition influence reproductive neuroendocrine and appetite responses to intracerebroventricular insulin and neuropeptide-Y in sheep.

Long-term nutritional background is thought to influence hypothalamic appetite and reproductive neuroendocrine responses to short-term nutritional feedback. In order to investigate this phenomenon, the effects of intracerebroventricular administration of insulin or neuropeptide-Y (NPY) on LH secretion and voluntary food intake (VFI) were examined in sheep that were initially thin and kept on an increasing nutritional plane (INP), or initially fat and kept on a decreasing nutritional plane (DNP), for 10 weeks. Intracerebroventricular insulin stimulated LH secretion and suppressed VFI in INP sheep when initially thin, but not when they became fat, and had no effect on LH in DNP sheep when initially fat, and stimulated LH secretion when they became thin. Intracerebroventricular NPY had no effect on LH or VFI in INP sheep when initially thin, decreased LH secretion and increased VFI when they became fat, and decreased LH secretion in DNP sheep when initially fat but had no effect when they became thin. Therefore, sensitivity to insulin increases with low or decreasing nutritional status and decreases with high or increasing nutritional status, whereas sensitivity to NPY increases with high or increasing nutritional status and decreases with low or decreasing nutritional status. In conclusion, reproductive neuroendocrine and appetite responses to acute changes in nutritional feedback signals depend on the individual's longer-term nutritional background.

Reduced content of α-synuclein in peripheral blood leukocytes of patients with LRRK2-associated Parkinson's disease.

Measurement of α-synuclein level in the peripheral blood was proposed as a diagnostic test for Parkinson's disease. However, the results of these studies remain contradictory, probably because the examined samples included patients with different etiology of Parkinson's disease. To verify this assumption we studied the levels of α-synuclein in peripheral blood leukocytes of patients with Parkinson's disease associated with mutations in the gene of leucine-rich kinase 2 (LRRK2). The mean α-synuclein level was significantly lower in patients with LRRK2-associated Parkinson's disease (N=8) than in patients with sporadic form of the disease (N=33; p<0.02) and in controls (N=18; p<0.05). On the other hand, we found no differences in the level of α-synuclein level between patients with sporadic form of the disease and controls. We hypothesize that the level of α-synuclein in the peripheral blood largely depends on the etiology of the disease and cannot be used as a universal diagnostic test for Parkinson's disease.

Percutaneous permeation comparison of repellents picaridin and DEET in concurrent use with sunscreen oxybenzone from commercially available preparations.

Concurrent application of insect repellent picaridin or DEET with sunscreens has become prevalent due to concerns on West Nile virus and skin cancer. The objectives of this study were to characterize the percutaneous permeation of picaridin and sunscreen oxybenzone from commercially available preparations and to compare the differences in permeability between picaridin and DEET in association with oxybenzone. In vitro diffusion studies were carried out to measure transdermal permeation of picaridin and oxybenzone from four different products, using various application concentrations and sequences. Results were then compared to those of repellent DEET and sunscreen oxybenzone under identical conditions. Transdermal permeation of picaridin across human epidermis was significantly lower than that of DEET, both alone and in combination with oxybenzone. Concurrent use resulted in either no changes or suppression of transdermal permeation of picaridin and oxybenzone. This finding was different from concurrent use of DEET and oxybenzone in which a synergistic permeation enhancement was observed. In addition, permeation of picaridin, DEET and oxybenzone across human epidermis was dependent on application concentration, use sequence, and preparation type. It was concluded from this comparative study that picaridin would be a better candidate for concurrent use with sunscreen preparations in terms of minimizing percutaneous permeation of the chemicals.

Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo.

Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1-deleted recombinant adenoviruses limit the duration of transgene expression.

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.

Metabolism of 6-methylbenz[a]anthracene by rat liver microsomes and mutagenicity of metabolites.

6-Methylbenz[a]anthracene (6-MBA) is metabolized by rat liver microsomes to form 3-hydroxy-6-MBA, 4-hydroxy-6-MBA, 5-hydroxy-6-MBA, 6-MBA trans-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols, and 4-hydroxy-6-MBA trans-10,11-dihydrodiol as the identifiable metabolites. 6-Hydroxymethylbenz[a]anthracene and its phenolic and dihydrodiol metabolites are also formed. The unique metabolites identified in 6-MBA metabolism are 6-MBA trans-5,6-dihydrodiol and 4-hydroxy-6-MBA trans-10,11-dihydrodiol. Metabolites were isolated by reversed-phase and normal-phase high-performance liquid chromatographies and identified by UV-visible absorption, mass, and proton nuclear magnetic resonance spectral analyses. Metabolites formed by low and high concentrations of liver microsomal enzymes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated male Sprague-Dawley rats were quantified by using [3H]6-MBA, with the tritium labeled at the methyl carbon, and liquid scintillation counting of fractions collected from reversed-phase high-performance liquid chromatography. Metabolic formations of 6-hydroxymethylbenz[a]anthracene, 6-MBA trans-dihydrodiols, and 4-hydroxy-6-MBA trans-10,11-dihydrodiol are highly dependent on the contents of cytochrome P-450 isozymes present in liver microsomes. The relative mutagenic activities of metabolites toward Salmonella typhimurium TA100 are: 6-MBA trans-3,4-dihydrodiol greater than 6-MBA trans-8,9-dihydrodiol greater than 6-MBA greater than 6-MBA trans-10,11-dihydrodiol greater than 4-hydroxy-6-MBA congruent to 4-hydroxy-6-MBA trans-10,11-dihydrodiol. The relatively high mutagenic activities of 6-MBA trans-3,4-dihydrodiol and 6-MBA trans-8,9-dihydrodiol suggest that both 6-MBA trans-3,4-dihydrodiol 1,2-epoxide(s) and 6-MBA trans-8,9-dihydrodiol 10,11-epoxide(s) may be the major metabolites which contribute to the carcinogenic properties of 6-MBA.

Identification and characterization of human beta 2-chimaerin: association with malignant transformation in astrocytoma.

Molecular processes resulting in the malignant transformation from low- to high-grade astrocytoma remain poorly understood. Using reverse transcriptase PCR, we identified a gene that is differentially expressed in normal brain and low-grade astrocytoma compared to glioblastoma tissues. This gene is identical to human beta 2-chimaerin, which encodes a 468-amino acid GTPase-activating protein for p21rac. The gene was localized to human chromosome 7p15.3 by fluorescence in situ hybridization mapping. Human beta 2-chimaerin is expressed in a variety of human tissues, with the highest expression level detected in human brain and pancreas. RNase protection assays indicated that the expression level of this gene is high in all the normal brain and low-grade astrocytoma samples tested compared to malignant gliomas. The down-regulation of beta 2-chimaerin expression in the high-grade gliomas suggests that decreased expression of this gene may be a feature of progression in the development of malignant glioma.

Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats.

Serum albumin was isolated from rats at 27 h after administration of the carcinogen [2,2'-3H]-4-aminobiphenyl. Pronase digestion of the purified albumin yielded a mixture of radiolabeled materials which was resolved into 5 major components by reverse-phase liquid chromatography. From detailed UV, 1H-NMR, and mass spectral analyses, four of these were determined to be 4-aminobiphenyl, 4'-hydroxy-4-acetylaminobiphenyl, and two other metabolites, all of which are presumed to be non-covalently associated with the serum albumin. The fifth component, however, resulted from covalent bond formation and was identified as a tetrapeptide containing 3-tryptophanyl-4-acetylaminobiphenyl, the amino acid sequence of which was H2N-ala-trp-ala-val. Since rat serum albumin contains only a single tryptophan residue in a hydrophobic drug binding site, its high selectivity for carcinogen binding suggests a unique role for this protein in the detoxification and/or transport of ultimate carcinogenic metabolites.

Sex Differences in Defensive Behavior and Venom of The Striped Bark Scorpion Centruroides vittatus (Scorpiones: Buthidae).

Studies of venom variability have advanced from describing the mechanisms of action and relative potency of medically important toxins to understanding the ecological and evolutionary causes of the variability itself. While most studies have focused on differences in venoms among taxa, populations, or age-classes, there may be intersexual effects as well. Striped bark scorpions (Centruroides vittatus) provide a good model for examining sex differences in venom composition and efficacy, as this species exhibits dramatic sexual dimorphism in both size and defensive behavior; when threatened by an enemy, larger, slower females stand and fight while smaller, fleeter males prefer to run. We here add evidence suggesting that male and female C. vittatus indeed have different defensive propensities; when threatened via an electrical stimulus, females were more likely to sting than were males. We reasoned that intersexual differences in defensive phenotypes would select for venoms with different functions in the two sexes; female venoms should be effective at predator deterrence, whereas male venoms, less utilized defensively, might be better suited to capturing prey or courting females. This rationale led to our predictions that females would inject more venom and/or possess more painful venom than males. We were wrong. While females do inject more venom than males in a defensive sting, females are also larger; when adjusted for body size, male and female C. vittatus commit equal masses of venom in a sting to a potential enemy. Additionally, house mice (Mus musculus) find an injection of male venom more irritating than an equal amount of female venom, likely because male venom contains more of the toxins that induce pain. Taken together, our results suggest that identifying the ultimate causes of venom variability will, as we move beyond adaptive storytelling, be hard-won.

Enzyme specificity under dynamic control: a normal mode analysis of alpha-lytic protease.

We have used alpha-lytic protease as a model system for exploring the relationship between the internal dynamics of an enzyme and its substrate specificity. The wild-type enzyme is highly specific for small substrates in its primary specificity pocket, while the M190A mutant has a much broader specificity, efficiently catalyzing cleavage of both large and small substrates. Normal modes have been calculated for both the wild-type and the mutant enzyme to determine how internal vibrations contribute to these contrasting specificity profiles. We find that for the atoms lining the walls of the specificity pocket, the wild-type normal modes have a more symmetric character, with the walls vibrating in phase, and the size of the pocket remaining relatively fixed. This is in agreement with X-ray crystallographic data on conformational substates trapped at 120 K. In contrast, we find that in the mutant, the binding pocket normal modes have a more antisymmetric character, with the walls vibrating out of phase, and the pocket able to expand and contract. These results suggest that the internal vibrations of a molecule may play an important role in determining substrate binding and specificity. A small change in protein structure can have a significant effect on the pattern of molecular vibrations, and thus on enzymatic properties, even if the overall amplitudes of the vibrations, as measured by NMR relaxation or crystallographic B-factors, remain largely unchanged.

Brain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a nuclear factor kappa B (NF-kappaB) signal transduction pathway.

The involvement of nuclear factor kappa B (NF-kappaB) in TNF-induced increases in cerebral microvascular permeability was evaluated both in vitro, using primary cultured bovine brain microvessel endothelial cells (BBMEC), and in vivo, using the rat cranial window model. In primary cultured BBMEC, TNF exposure resulted in an increased appearance of the Rel A subunit of NF-kappaB in immunoblots of cell lysates. Increases in the Rel A subunit of NF-kappaB were observed as early as 30-min after administration of TNF. The increased permeability and the secretion of prostaglandin E2 in response to TNF exposure in BBMEC monolayers were significantly reduced by several different NF-kappaB inhibitors, including PDTC, CAPE, BAY 11-7085, and lactacystin. Similar results were also obtained in the rat cranial window model where treatment with the COX-2 inhibitor, NS-398 (0.1 microM), or the NF-kappaB inhibitor, PDTC (10 microM), significantly reduced the permeability increases produced by TNF. These studies suggest that the increases in BBB permeability following TNF exposure are attributable to activation of an NF-kappaB-mediated signaling pathway in the cerebral microvasculature.

A clinical interactive technique for MR-CT image registration for target delineation of intracranial tumors.

Replacement of current CT-based, three-dimensional (3D) treatment planning systems by newer versions capable of automated multi-modality image registration may be economically prohibitive for most radiation oncology clinics. We present a low-cost technique for MR-CT image registration on a "first generation" CT-based, 3D treatment planning system for intracranial tumors. The technique begins with fabrication of a standard treatment mask. A second truncated mask, the "minimask," is then made, using the standard mask as a mold. Two orthogonal leveling vials glued onto the minimask detect angular deviations in pitch and roll. Preservation of yaw is verified by referencing a line marked according to the CT laser on the craniocaudal axis. The treatment mask immobilizes the patient's head for CT. The minimask reproduces this CT-based angular treatment position, which is then maintained by taping the appropriately positioned head to the MR head coil for MR scanning. All CT and MR images, in DICOM 3.0 format, are entered into the treatment planning system via a computer network. Interactive registration of MR to CT images is controlled by real-time visual feedback on the computer monitor. Translational misalignments at the target are eliminated or minimized by iterative use of qualitative visual inspection. In this study, rotational errors were measured in a retrospective series of 20 consecutive patients who had undergone CT-MR image registration using this technique. Anatomic structures defined the three CT orthogonal axes from which angular errors on MR image were measured. Translational errors at the target isocenter were within pixel size, as judged by visual inspection. Clinical setup using the minimask resulted in overall average angular deviation of 3 degrees +/-2 degrees (mean +/- SD) and translational deviation within the edges of the target volume of typically less than 2 mm. The accuracy of this registration technique for target delineation of intracranial tumors is compatible with practice guidelines. This method, then, provides a cost-effective means to register MR and CT images for target delineation of intracranial tumors.

T cell adoptive immunotherapy of newly diagnosed gliomas.

Patients with newly diagnosed gliomas were treated with adoptive transfer of ex vivo activated T lymphocytes, derived from lymph nodes (LNs) draining autologous tumor vaccines, to determine the long-term toxicity of this treatment. Twelve consecutive patients were enrolled: 2 with grade II astrocytoma, 4 with anaplastic gliomas, and 6 with glioblastoma multiforme. Patients were injected intradermally with short-term cultured autologous irradiated tumor cells, admixed with granulocyte macrophage colony-stimulating factor, to stimulate draining LNs. The LN cells were activated with staphylococcal enterotoxin A for 48 h and then cultured in medium containing interleukin 2 for an additional 6-8 days and subsequently transferred i.v. to the patients. The number of cells obtained from the LNs ranged from 9 x 10(7) to 1.1 x 10(9), and the median cell proliferation was 41-fold. The dose of T cells infused ranged from 0.6 to 5.5 x 10(10) with a median of 1.1 x 10(10), the majority of which were CD 4+ (mean, 71%). The entire treatment was performed as outpatient therapy and was associated with a toxicity of grade 2 or less, consisting mainly of fever, nausea, and myalgias during the first 24 h. There were no indications of late adverse events from this treatment even among three patients with follow-up greater than 2 years post T cell transfer. Moreover, four patients demonstrated partial regression of residual tumor. This Phase I clinical trial of adoptive immunotherapy for patients with newly diagnosed malignant gliomas demonstrates feasibility, lack of long-term toxicity, and several objective clinical responses.

Benefits of coronary artery bypass surgery.

More than 150,000 Americans have undergone coronary artery bypass surgery since this operation was first available on a nationwide scale in 1968. From data now available, we can better evaluate the various mechanisms by which bypass surgery has been thought to relieve anginal symptoms, and whether or not this operation improves ventricular function and increases survival. Most individuals have substantial or complete relief of angina after surgery, no longer require nitroglycerin or beta blocking agents, and are able to return to full physical and sexual activity. A bypass graft restores the capacity of a proximally obstructed coronary artery to increase its blood flow level up to fourfold more than resting levels with exertion. Also, evidence is now appearing that bypass surgery can improve survival in patients with advanced coronary artery disease.

A statistical mechanical model for hydrogen exchange in globular proteins.

We develop a statistical mechanical theory for the mechanism of hydrogen exchange in globular proteins. Using the HP lattice model, we explore how the solvent accessibilities of chain monomers vary as proteins fluctuate from their stable native conformations. The model explains why hydrogen exchange appears to involve two mechanisms under different conditions of protein stability: (1) a "global unfolding" mechanism by which all protons exchange at a similar rate, approaching that of the denatured protein, and (2) a "stable-state" mechanism by which protons exchange at rates that can differ by many orders of magnitude. There has been some controversy about the stable-state mechanism: does exchange take place inside the protein by solvent penetration, or outside the protein by the local unfolding of a subregion? The present model indicates that the stable-state mechanism of exchange occurs through an ensemble of conformations, some of which may bear very little resemblance to the native structure. Although most fluctuations are small-amplitude motions involving solvent penetration or local unfolding, other fluctuations (the conformational distant relatives) can involve much larger transient excursions to completely different chain folds.

Ligand binding to proteins: the binding landscape model.

Models of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand-induced denaturation, ANS and heme-like binding, and "lock-and-key" and "induced-fit" specific binding behaviors characterized by Michaelis-Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis-Menten isotherms. Even low-population binding to denatured states can cause changes in global stability, hydrogen-exchange rates, and thermal B-factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass-action models.

Development of a hospital-based proton beam treatment center.

Radiation oncologists recognize a continuing need to improve the radiation dose distribution between a cancer and the surrounding normal tissue. A most promising method of accomplishing this goal is the use of charged particle beam irradiation, the clinical use of which has been investigated for the past 40 years. Since the first clinical studies began at the Lawrence Berkeley Laboratory in 1954, more than 5,000 patients have been treated with protons, using accelerators designed for physics laboratories. Superior results are reported for the control of selected diseases by the ten facilities which are currently investigating proton radiation therapy. The findings have resulted in expansion plans in several of these facilities, and in the formulation of plans for two new facilities. We report on the planned development of a new facility at Loma Linda University, which has contracted with Fermi National Accelerator Laboratory for the design and fabrication of a 250 MeV synchrotron and its beam transport and delivery systems. This facility will be the first in the world to employ a proton accelerator dedicated to medical service and research. As such, it will be available as an international resource to develop and improve the modality.

Methodologies and tools for proton beam design for lung tumors.

PURPOSE: Proton beams can potentially increase the dose delivered to lung tumors without increasing the dose to critical normal tissues because protons can be stopped before encountering the normal tissues. This potential can only be realized if tissue motion and planning uncertainties are correctly included during planning. This study evaluated several planning strategies to determine which method best provides adequate tumor coverage, minimal normal tissue irradiation, and simplicity of use. METHODS AND MATERIALS: Proton beam treatment plans were generated using one or more of three different planning strategies. These strategies included designing apertures and boluses to the PTV, apertures to the PTV and boluses to the CTV, and aperture and bolus to the CTV. RESULTS: The planning target volume as specified in ICRU Report 50 can be used only to design the lateral margins of beams, because the distal and proximal margins resulting from CT number uncertainty, beam range uncertainty, tissue motions, and setup uncertainties, are different than the lateral margins resulting from these same factors. The best strategy for target coverage with the planning tools available overirradiated some normal tissues unnecessarily. The available tools also made the planning of lung tumors difficult. CONCLUSIONS: This study demonstrated that inclusion of target motion and setup uncertainties into a plan should be performed in the beam design step instead of creating new targets. New computerized treatment planning system tools suggested by this study will ease planning, facilitate abandonment of the PTV concept, improve conformance of the dose distribution to the target, and improve conformal avoidance of critical normal tissues.

The proton treatment center at Loma Linda University Medical Center: rationale for and description of its development.

Proton radiation, a continuation of radiation oncology's historic search for an optimum dose distribution, offers superior characteristics for clinical radiation therapy. A complete facility for clinical proton radiation therapy has been designed for and constructed at Loma Linda University Medical Center. To bring about this achievement, a consortium of engineers, physicists, and physicians interested in the clinical applications of protons was necessary. The accelerator, the beam transport and delivery systems, the building, and the personnel who operate the system were all brought together to fully exploit the properties of protons for patient treatments, which are now underway.