A noncalibration spectroscopic method to estimate ether extract and fatty acid digestibility of feed and its validation with flaxseed and field pea in pigs.

Digestibility of ether extract (EE) or fatty acids (FA) is traditionally measured by chemical analyses for EE or GLC methods for FA combined with marker concentration in diet and digesta or feces. Digestibility of EE or FA may be predicted by marker concentrations and spectral analyses of diet and digesta or feces. On the basis of Beer's law, a noncalibration spectroscopic method, which used functional group digestibility (FGD) determined with marker concentration and peak intensity of spectra of diets and undigested residues (digesta or feces), was developed to predict the apparent ileal digestibility (AID) of total FA and apparent total tract digestibility (ATTD) of EE. To validate, 4 diets containing 30% flaxseed and field pea coextruded with 4 extruder treatments and a wheat and soybean basal diet with predetermined AID of total FA and ATTD of EE were used. Samples of ingredients, diets, and freeze-dried digesta and feces were scanned on a Fourier transform infrared (FT-IR) instrument with a single-reflection attenuated total reflection (ATR) accessory. The intensity of either the methylene (CH2) antisymmetric stretching peak at 2,923 cm(-1) (R(2) = 0.90, P < 0.01) or the symmetric stretching peak at 2,852 cm(-1) (R(2) = 0.86, P < 0.01) of ingredients, diet, and digesta spectra was related strongly to the concentration of total FA. The AID of total FA of diets measured using GLC was predicted by the spectroscopic method using FGD at 2,923 and 2,852 cm(-1) (R(2) = 0.75, P < 0.01) with a bias of 0.54 (SD = 3.78%) and -1.35 (SD = 3.74%), respectively. The accumulated peak intensity in the region between 1,766 and 1,695 cm(-1) of spectra was related to EE concentration in ingredients and diets (R(2) = 0.61, P = 0.01) and feces (R(2) = 0.88, P < 0.01). The relation was improved by using second-derivative spectra of the sum of peak intensities at 1,743 and 1,710 cm(-1) for ingredients and diets (R(2) = 0.90, P = 0.01) and at 1,735 and 1,710 cm(-1) for feces (R(2) = 0.92, P < 0.01). The ATTD of EE of test diets determined with proximate analysis was estimated by the FGD of nonderivative spectra with or without baseline (R(2) = 0.90, P < 0.01) with a bias of 3.15 (SD = 3.14%) and 3.50 (SD = 3.24%), respectively. In conclusion, instead of using GLC methods or predictions based on calibrations, the AID of total FA and ATTD of EE can also be estimated directly from ATR FT-IR spectra, provided the ratio of marker in the diet and undigested residue is known.

Three-dimensional structure of Bax-mediated pores in membrane bilayers.

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ~3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.

p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Age-related alterations in the inflammatory response to dermal injury.

Previous studies have documented that the ability to heal wounds declines with age. Although many factors contribute to this age-associated deficit, one variable that has not been carefully examined is leukocyte recruitment and function in wounds. This investigation compares the inflammatory response in excisional wounds of young (age 8 wk) and aged (age 22 mo) mice. In the early inflammatory response, neutrophil content of wounds was similar for both aged and young mice. In contrast, macrophage levels were 56% higher in aged versus young mice (81 +/- 20 vs 52 +/- 13 cells per mm2). In the later inflammatory response, wounds of aged mice exhibited a delay in T cell infiltration, with maximum T cell levels at day 10 in aged mice versus day 7 in young mice. Despite this delay, the eventual peak concentration of T cells was 23% higher in the wounds of aged mice (152 +/- 11 cells per mm2 vs 124 +/- 21cells per mm2). The observed alterations in inflammatory cell content suggested that chemokine production might be altered with age. An elevation of monocyte chemoattractant protein (MCP-1) levels was observed in wounds of aged mice. RNase protection studies, however, revealed that the production of most chemokines, including MIP-2, MIP-1alpha, MIP-1beta, and eotaxin, tended to decline with age. Because optimal wound healing requires both appropriate macrophage infiltration and phagocytic activity, phagocytosis was examined. Compared to young mice, wound macrophages from aged mice exhibited a 37%-43% reduction in phagocytic capacity. Taken together, the data demonstrate age-related shifts in both macrophage and T cell infiltration into wounds, alterations in chemokine content, and a concurrent decline in wound macrophage phagocytic function. These alterations may contribute to the delayed repair response of aging.

Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein.

Fibroblast growth factor-binding protein (FGF-BP) 1 is a secreted protein that can bind fibroblast growth factors (FGFs) 1 and 2. These FGFs are typically stored on heparan sulfate proteoglycans in the extracellular matrix in an inactive form, and it has been proposed that FGF-BP1 functions as a chaperone molecule that can mobilize locally stored FGF and present the growth factor to its tyrosine kinase receptor. FGF-BP1 is up-regulated in squamous cell, colon, and breast cancers and can act as an angiogenic switch during malignant progression of epithelial cells. For the present studies, we focused on FGF-1 and -2 and investigated interactions with recombinant human FGF-BP1 protein as well as effects on signal transduction, cell proliferation, and angiogenesis. We show that recombinant FGF-BP1 specifically binds FGF-2 and that this binding is inhibited by FGF-1, heparan sulfate, and heparinoids. Furthermore, FGF-BP1 enhances FGF-1- and FGF-2-dependent proliferation of NIH-3T3 fibroblasts and FGF-2-induced extracellular signal-regulated kinase 2 phosphorylation. Finally, in the chicken chorioallantoic membrane angiogenesis assay, FGF-BP1 synergizes with exogenously added FGF-2. We conclude that FGF-BP1 binds directly to FGF-1 and FGF-2 and positively modulates the biological activities of these growth factors.

Parotid and thyroid gland cancers in patients with ataxia-telangiectasia.

This study describes the clinicopathologic features of parotid and thyroid gland cancers in patients with ataxia-telangiectasia (AT). The medical records of 412 AT patients were reviewed to identify those patients who developed parotid or thyroid gland cancers. Presenting features, diagnoses, types of therapy, risk factors, and other primary cancers were analyzed. Five patients with parotid or thyroid gland cancers were identified. Three had parotid (2 mucoepidermoid and 1 acinic cell) and 2 had thyroid gland (1 papillary and 1 follicular) cancers. Four patients presented with head and neck masses and 1 had an occult papillary thyroid carcinoma. Four patients had more than one primary cancer. The only mode of therapy was surgery. The 2 patients with mucoepidermoid carcinoma had complete parotidectomies. One is alive without any evidence of disease 12 months after diagnosis and 1 died of refractory lymphoma without any evidence of mucoepidermoid carcinoma at autopsy. The patient with acinic cell carcinoma had a parotid biopsy only. The 2 patients with thyroid cancer were diagnosed at autopsy. The results indicate that patients with AT are at risk for developing multiple primary cancers including those of the parotid and thyroid gland, and should be evaluated for such primaries.

Mortality rates among carriers of ataxia-telangiectasia mutant alleles.

BACKGROUND: Mutations at the ataxia-telangiectasia locus cause a distinctive autosomal recessive syndrome in homozygotes and predispose heterozygotes to cancer and ischemic heart disease. OBJECTIVE: To examine mortality rates among persons carrying a mutated ataxia-telangiectasia gene. DESIGN: Retrospective cohort study. SETTING: The United States and Canada. PARTICIPANTS: 405 grandparents of patients with ataxia-telangiectasia. MEASUREMENTS: Ages at death and risk for death (from all causes, cancer, ischemic heart disease, and other causes) among carriers and noncarriers of ataxia-telangiectasia mutations. RESULTS: Compared with noncarriers, carriers of a mutated ataxia-telangiectasia allele had a significantly increased risk for death at 20 through 79 years of age (relative risk, 1.9 [95% CI, 1.3 to 2.8]) (P < 0.001). On average, carriers died 7 to 8 years earlier than noncarriers. Cancer caused most of the excess deaths, and ischemic heart disease caused the remainder. Among carriers, relative risk for death from cancer and ischemic heart disease before 80 years of age was 2.6 (CI, 1.4 to 4.7; P = 0.002) and 2.0 (CI, 1.0 to 4.0; P = 0.062), respectively. Compared with noncarriers, carriers who died of cancer were a mean of 4 years younger (P > 0.2) and carriers who died of ischemic heart disease were a mean of 11 years younger (P = 0.006). CONCLUSION: Carriers of mutations at the ataxia-telangiectasia locus, who make up 1.4% to 2% of the general population, have a higher mortality rate and an earlier age at death from cancer and ischemic heart disease than noncarriers.

Mutations at the ataxia-telangiectasia locus and clinical phenotypes of A-T patients.

Mutations at the ataxia-telangiectasia (A-T) locus on chromosome band 11q22 cause a distinctive autosomal recessive syndrome in homozygotes and predispose heterozygotes to cancer, ischemic heart disease, and early mortality. PCR amplification from genomic DNA and automated sequencing of the entire coding region (66 exons) and splice junctions detected 77 mutations (85%) in 90 A-T chromosomes. Heteroduplex analysis detected another 42 mutations at the A-T locus. Out of a total of 71 unique mutations, 50 were found only in a single family, and 51 had not been reported previously. Most (58/71, 82%) mutations were frameshift and nonsense mutations that are predicted to cause truncation of the A-T protein; the less common mutation types were missense (9/71, 13%), splicing (3/71, 4%) and one in-frame deletion, 2546 3 (1/71, 1%). The mean survival and height distribution of 134 A-T patients correlated significantly with the specific mutations present in the patients. Patients homozygous for a single truncating mutation, typically near the N-terminal end of the gene, or heterozygous for the in-frame deletion 2546 3, were shorter and had significantly shorter survival than those heterozygous for a splice site or missense mutation, or heterozygous for two truncating mutations. Alterations of the length or amino acid composition of the A-T gene product affect the A-T clinical phenotype in different ways. Mutation analysis at the A-T locus may help estimate the prognosis of A-T patients.

Neutral sequence variants and haplotypes at the 150 Kb ataxia-telangiectasia locus.

Sequence variants occur every few hundred bases in the human genome. We evaluated the relationship between disease-causing mutations and neutral sequence variants at the 150 Kb ataxia-telangiectasia (A-T) locus. Mutations at this locus cause a distinct autosomal recessive syndrome in homozygotes and predispose heterozygotes to cancer and coronary heart disease. Nine common neutral sequence variants were observed in the coding and splice junction regions of 132 chromosomes from Caucasian individuals of European origin. Each of these variants appeared frequently in both A-T and non-A-T chromosomes. However, there was remarkable linkage disequilibrium between the polymorphic loci, resulting in only 7 haplotypes in analyzed chromosomes. These 7 haplotypes fell into 3 major ancestral groups. No individual polymorphic variant or haplotype correlated reliably with the presence of an A-T mutation. Thus, comparing the frequency of neutral variants at the A-T locus in diseased and non-diseased populations is unlikely to uncover the relationship of mutations at this locus to common diseases. These data reflect general limitations on using single nucleotide polymorphisms (SNPs) to identify loci for many common diseases.

Impaired wound repair and delayed angiogenesis in aged mice.

Wound repair is a multistep process consisting of hemostasis, inflammatory cell infiltration, tissue regrowth, and remodeling. In aged individuals, this progression of events is altered, resulting in wounds that heal more slowly than wounds in the young. These studies were designed to examine the proliferative phase of repair in young and aged mice, with attention to the angiogenic process. Using a standardized excisional injury model, wound re-epithelialization, collagen accumulation, and angiogenesis were examined. Re-epithelialization and collagen synthesis were substantially delayed in aged mice as compared with young mice. Angiogenesis in wounds from aged mice was also delayed, with significantly more capillary growth in wounds from young mice than aged mice. In addition, wounds from aged mice contained significantly less of the angiogenic mediators fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) than wounds from young animals (p < 0.05). Because macrophages are a rich source of angiogenic factors in wounds, macrophage production of VEGF was examined. Macrophages from aged mice produced significantly less VEGF than cells from young mice. To examine the in vivo endothelial cell responsiveness, a defined amount of rFGF-2 was suspended in Matrigel and placed subcutaneously in either young or aged mice. In response to FGF-2, capillary growth into Matrigel was significantly less in aged than young mice. The results suggest that a decline in angiogenic growth factor production, as well as a decline in endothelial responsiveness to specific factors, may account for the delayed wound angiogenesis in aged mice. These results also indicate that age-related alterations in macrophage function might partially account for the overall delay in the wound repair process.

Treatment of lymphoid malignancies in patients with ataxia-telangiectasia.

BACKGROUND: Patients with ataxia-telangiectasia (A-T) are at an increased risk for developing lymphoid malignancies, yet the appropriate therapy remains unknown. Radiation therapy at conventional doses results in destruction of normal tissue, which has suggested that full-dose chemotherapy might result in unacceptable toxicity in A-T patients with cancer. PROCEDURE: The medical records of 412 A-T patients were reviewed to identify those patients who developed lymphoid malignancies and to analyze the type and duration of therapy, events during therapy, and off-therapy follow-up. RESULTS: Of 74 A-T patients with lymphoid malignancies, 32 patients received chemotherapy. The 21 patients treated with standard chemotherapy had a significantly better median survival (9 months, range, 1-162+ months vs. 5 months, range, 0.5-28 months) (P = 0.03) and complete remission rate (76% vs. 9%) (P = 0.001) than the 11 treated with reduced dose chemotherapy. Three of the 21 full-dose chemotherapy patients required dose reductions because of neutropenia. Seven of the 14 patients exposed to 1,200 mg/m2 or greater of cyclophosphamide developed hemorrhagic cystitis. All three patients exposed to bleomycin developed pulmonary disease which was fatal in two. Of the 16 standard-dose chemotherapy patients who achieved a complete remission, two remain disease-free, five have died of recurrent disease, and five died of pulmonary disorders and four of other causes while in remission. CONCLUSIONS: Standard-dose chemotherapy should be given to each A-T patient with a lymphoid malignancy unless additional physical or emotional problems make it unlikely that the patient will benefit. Morbidity and mortality may be reduced by prophylaxis against hemorrhagic cystitis and early detection and treatment of pulmonary disorders.

Radiosensitivity of normal tissues in ataxia-telangiectasia heterozygotes.

PURPOSE: Approximately 5% of cancer patients given radiation therapy exhibit severe injuries to the noncancerous tissue in the radiation field. Striking clinical sensitivity to ionizing radiation has been observed frequently in ataxia-telangiectasia (A-T) homozygotes. This study was undertaken to test the hypothesis that heterozygous carriers of a mutated gene for A-T may represent a substantial proportion of all patients who suffer severe radiation toxicity. METHODS: The medical records of all A-T heterozygotes treated with radiation therapy for breast or prostate cancer were compiled from an ongoing study of mortality and cancer incidence in A-T families. Diagnostic, treatment, and follow-up records were reviewed. Acute and long-term radiation complications were scored according to Radiation Therapy and Oncology Group criteria. RESULTS: There were no instances of soft tissue necrosis or other apparent serious injuries to normal tissues of two A-T heterozygotes with prostate carcinoma and 11 with breast carcinoma who received moderate-to-high doses of conventionally fractionated radiation therapy by megavoltage techniques. CONCLUSION: There is no evidence that abnormal clinical radiosensitivity occurs in A-T heterozygotes receiving conventionally fractionated radiation therapy for breast or prostate cancer.

Giardia and Cryptosporidium in dairy calves in British Columbia.

A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive.