Effects of dietary fish meal and soybean meal on the ovine innate and acquired immune response during pregnancy and lactation.

In recent years, livestock producers have been supplementing animal diets with fish meal (FM) to produce value-added products for health conscious consumers. As components of FM have unique neuroendocrine-immunomodulatory properties, we hypothesize that livestock producers may be influencing the overall health of their animals by supplementing diets with FM. In this study, 40 pregnant ewes were supplemented with rumen protected (RP) soybean meal (SBM: control diet) or RP FM, commencing gestation day 100 (gd100), in order to evaluate the impact of FM supplementation on the innate and acquired immune response and neuroendocrine response of sheep during pregnancy and lactation. On gd135, half the ewes from each diet (n = 10 FM, n = 10 SBM) were challenged iv with lipopolysaccharide (LPS) to simulate a systemic bacterial infection and the febrile, respiratory and neuroendocrine responses were monitored over time; the other half (n = 10 FM, n = 10 SBM) of the ewes received a saline injection as control. On lactation day 20 (ld20), all ewes (n = 20 FM, n = 20 SBM) were sensitized with hen egg white lysozyme (HEWL) and the serum haptoglobin (Hp) response was measured over time. The cutaneous hypersensitivity response (CHR) to HEWL challenge was measured on ld30 (n = 20 FM, n = 20 SBM), and blood samples were collected over time to measure the primary and secondary immunoglobulin G (IgG) response to HEWL. There was an attenuated trend in the LPS-induced febrile response by the FM treatment when compared with the SBM treatment (P = 0.06), as was also true for the respiratory response (P = 0.07), but significant differences in neuroendocrine function (serum cortisol and plasma ACTH) were not observed between treatments. Basal Hp levels were significantly lower in the FM supplemented ewes when compared with the SBM supplemented ewes (P < 0.01), and the Hp response to HEWL sensitization differed significantly over time between treatments (P < 0.01). The CHR to HEWL was also significantly attenuated in the FM treatment compared with the SBM (P < 0.01); however, treatment differences in the primary and secondary IgG responses to HEWL were not observed. These results indicate that FM supplementation differentially affects the innate and acquired immune responses in pregnant and lactating sheep compared with a typical SBM diet of commercial flocks. The long-term implications of this immunomodulation warrant further investigation.

3D-RISM-KH molecular theory of solvation and density functional theory investigation of the role of water in the aggregation of model asphaltenes.

We applied a multiscale modeling approach that involves the statistical-mechanical three-dimensional reference interaction site model with the Kovalenko-Hirata closure approximation (3D-RISM-KH molecular theory of solvation) as well as density functional theory (DFT) of electronic structure to study the role of water in aggregation of the asphaltene model compound 4,4'-bis(2-pyren-1-yl-ethyl)-2,2'-bipyridine (PBP) [X. Tan, H. Fenniri and M. R. Gray, Energy Fuels, 2008, 22, 715]. The solvation free energy and potential of mean force predicted by 3D-RISM-KH reveal favorable pathways for disaggregation of PBP dimers in pure versus water-saturated chloroform solvent. The water density distribution functions elucidate hydrogen bonding preferences and water bridge formation between PBP monomers. The ΔG(298) values of -5 to -7 kcal mol(-1) for transfer of water molecules in chloroform to a state interacting with PBP molecules are in agreement with experimental results. Geometry optimization and thermochemistry analysis of PBP dimers with and without water bridges using WB97Xd/6-31G(d,p) predict that both PBP dimerization and dimer stabilization by water bridges are spontaneous (ΔG(298) < 0). The (1)H NMR chemical shifts of PBP monomers and dimers predicted using the gauge-independent atomic orbital method and polarizable continuum model for solvation in chloroform are in an excellent agreement with the experimental results for dilute and concentrated PBP solutions in chloroform, respectively [X. Tan, H. Fenniri and M. R. Gray, Energy Fuels, 2009, 23, 3687]. The DFT calculations of PBP dimers with explicit water show that bridges containing 1-3 water molecules lead to stabilization of PBP dimers. Additional water molecules form hydrogen bonds with these bridges and de-shield the PBP protons, negating the effect of water on the (1)H(C3) NMR chemical shift of PBP, in agreement with experiment. The ΔG(298) results show that hydrogen bonding to water and water-promoted polynuclear assembly bridging is as important as π-π interactions for asphaltene aggregation.

Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.

Survival following extended field irradiation in carcinoma of cervix metastatic to para-aortic lymph nodes.

OBJECTIVE: Our goal was to determine survival after extended-field treatment of para-aortic lymph node (PALN) metastasis. METHODS: Thirty-five patients were treated from 1975-1989 for PALN metastasis. The FIGO stages were IB 10, 2A 3, IIB 9, IIIA 1, IIIB 10, 4A 1, and unstaged 1. The diagnosis in 34 patients was by operative staging and in 1 by CT scan and fine-needle aspiration biopsy. Twelve patients had microscopic PALN metastasis (PALN1) and 23 had grossly enlarged lymph nodes (PALN2). Thirty-four patients had extended-field radiotherapy (RT) plus brachytherapy or pelvic boost. Kaplan-Meier estimates were computer calculated for the entire population. Late radiation morbidity was classified by RTOG/EORTC criteria. RESULTS: The 5-year overall survival rate was approximately 29%. Four patients (3 stage IB, 1 stage IIIA) survived without recurrence. All four had extended field RT. The 5-year survival rate was 41.7% for PALN1 cases and 26.1% for PALN2 cases. Three patients (8.6%) had Grade 4 morbidity. CONCLUSIONS: PALN metastasis in stage IB is curable in approximately 30% of cases. The management approach in this series in stage IB was as follows: If PALN metastasis was identified at exploration for radical hysterectomy, the procedure was aborted and extended-field RT administered. In stages IIB through IVA, operative staging or CT scanning with FNA biopsy of suspicious PALN was performed. If PALN metastasis was confirmed, extended-field RT was administered. A 35% 5-year survival rate was observed in the advanced group. The value of chemotherapy for PALN metastasis remains to be defined but results from clinical trials suggest that cisplatin-based chemotherapy may be beneficial.

The right end of the vir region of an octopine-type Ti plasmid contains four new members of the vir regulon that are not essential for pathogenesis.

We sequenced the virD-virE, virE-virF, and virF-T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the vir gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensable for tumorigenesis.

Radiotherapy for vaginal carcinoma: a 23-year review.

The purpose of the study was to evaluate prognostic variables and morbidity in patients with vaginal carcinoma. 34 patients, mean age 67 years (+/- 8 SD), were treated between 1976 and 1994. 14 patients had a history of prior hysterectomy. In 13 of the 34 patients the tumour site was the upper vagina, in 9 it was the middle third, in 8 the lower third and in 4 the entire length of the vagina. Disease stage was I in 9 patients, II in 16, III in 7 and IV in 2. There were four treatment groups: external beam therapy + intracavitary brachytherapy (Group WPIC, n = 15); external beam therapy + interstitial brachytherapy (Group WPIS, n = 10); external beam therapy alone (Group WP, n = 7); and brachytherapy alone (Group BA, n = 2). Kaplan-Meier estimates and log-rank tests were used to evaluate survival. Disease-specific 5-year survival was 68% for 28 patients with squamous cell carcinoma and 50% for 6 patients with adenocarcinoma (p-value 0.3). 5-year survival was 78% for stage I disease, 63% for stage II, 33% for stage III and 50% for stage IV (p-value 0.2). Vaginal site of carcinoma, history of hysterectomy and treatment type are not significant prognostic factors. Local failure occurred in 2 patients (13%) in the WPIC group, 2 (20%) in WPIS, 3 (43%) in WP and 1 (50%) in BA. 9 patients (26%) had late small/large intestine and/or bladder morbidity. Vaginal morbidity occurred in 15 patients (44%); 9/15 (60%) in the WPIC group and 3/10 (30%) in the WPIS group having vaginal morbidity. This means that, when combining external beam therapy with brachytherapy, interstitial techniques are preferred over intracavitary techniques.

Assessment of accuracy of daily set-ups in prostate radiotherapy using electronic imaging.

The purpose of the study was to assess the accuracy, using electronic portal imaging, of daily set-ups, in patients undergoing radiotherapy for prostate carcinoma. We used a scanning liquid ion chamber to assess the accuracy of set-ups in 25 consecutive patients undergoing a 6 1/2 week course of radiotherapy to the prostate. Electronic images (EPIs) were collected during 33 treatments to each of four ports. The positions of anatomical structures on the EPIs were compared with the same structures seen on digitally reconstructed radiographs (DRRs) made after CT simulation before beginning radiotherapy. Displacements of the EPIs compared with the DRRs were computer-calculated in millimetres in lateral, longitudinal and rotational directions for each port. 11 patients had ports moved because of discrepancies between the EPIs and the DRRs; eight required moves in the first five treatments to correct systematic (simulator) errors. In the right-left and anterior-posterior directions, nearly 95% of the EPIs were within 5 mm of the simulated port position. In the superior-inferior direction, 98% of the ports were within 5 mm of the simulated port position. Two patients had in-plane rotational errors on the lateral ports (8 degrees and 10 degrees respectively). It was concluded that daily electronic imaging is an effective technique for assessing the accuracy of set-ups in prostate radiotherapy.

High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

A centromere DNA-binding protein from fission yeast affects chromosome segregation and has homology to human CENP-B.

Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y., J.A. Huberman, and J. Hurwitz. 1996. Proc. Natl. Acad. Sci. USA. 93:502-507). Abp1p binds in vitro specifically to at least three sites in centromeric central core DNA of S. pombe chromosome II (cc2). Overexpression of abp1 affects mitotic chromosome stability in S. pombe. Although inactivation of the abp1 gene is not lethal, the abp1 null strain displays marked mitotic chromosome instability and a pronounced meiotic defect. The identification of a CENP-B-related centromere DNA-binding protein in S. pombe strongly supports the hypothesis that fission yeast centromeres are structurally and functionally related to the centromeres of higher eukaryotes.

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates.

Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the Tral protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-Tral (H6-Tral) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H6-Tral used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.

Osteoradionecrosis of the hyoid induced by combined modality therapy for laryngeal carcinoma.

Contemporary management of laryngeal carcinoma often incorporates multiple modalities of therapy. We report a case of osteoradionecrosis of the hyoid bone in a patient treated with surgery, chemotherapy, and radiation therapy for a supraglottic squamous cell carcinoma. A discussion regarding pathophysiology, radiation dosimetry and treatment options is also presented.

Prevention of HIV infection. Looking back, looking ahead.

For some, the occurrence of as many as 40,000 new human immunodeficiency virus (HIV) infections in the United States each year is evidence that HIV education and prevention efforts have failed. To the contrary, more than a decade of experience with HIV has demonstrated that lasting changes in behavior needed to avoid infection can occur as a result of carefully tailored, targeted, credible, and persistent HIV risk-reduction efforts. Given experience in other health behavior change endeavors, no interventions are likely to reduce the incidence of HIV infection to zero; indeed, insisting on too high a standard for HIV risk-reduction programs may actually undermine their effectiveness. A number of social, cultural, and attitudinal barriers continue to thwart the implementation of promising HIV risk-reduction programs. The remote prospects for a successful prophylactic vaccine for HIV and the difficulty in finding effective drug treatments have underscored the importance of sustained attention to HIV prevention and education. A series of "correlates of immunity" are identified--precedents that must exist to establish effective HIV prevention programs. These include sound policies promoting HIV risk reduction; access to health and social services, condoms, needles, and syringes; interventions shown to motivate behavioral change; organizations capable of reaching those at risk; and development and diffusion of technologies to interrupt the spread of the virus.