[Development and validation of a prognostic prediction model for patients with stage â to â ¢ colon cancer incorporating high-risk pathological features].

Objective: To examine a predictive model that incorporating high risk pathological factors for the prognosis of stage Ⅰ to Ⅲ colon cancer. Methods: This study retrospectively collected clinicopathological information and survival outcomes of stage Ⅰ~Ⅲ colon cancer patients who underwent curative surgery in 7 tertiary hospitals in China from January 1, 2016 to December 31, 2017. A total of 1 650 patients were enrolled, aged (M(IQR)) 62 (18) years (range: 14 to 100). There were 963 males and 687 females. The median follow-up period was 51 months. The Cox proportional hazardous regression model was utilized to select high-risk pathological factors, establish the nomogram and scoring system. The Bootstrap resampling method was utilized for internal validation of the model, the concordance index (C-index) was used to assess discrimination and calibration curves were presented to assess model calibration. The Kaplan-Meier method was used to plot survival curves after risk grouping, and Cox regression was used to compare disease-free survival between subgroups. Results: Age (HR=1.020, 95%CI: 1.008 to 1.033, P=0.001), T stage (T3:HR=1.995,95%CI:1.062 to 3.750,P=0.032;T4:HR=4.196, 95%CI: 2.188 to 8.045, P<0.01), N stage (N1: HR=1.834, 95%CI: 1.307 to 2.574, P<0.01; N2: HR=3.970, 95%CI: 2.724 to 5.787, P<0.01) and number of lymph nodes examined (≥36: HR=0.438, 95%CI: 0.242 to 0.790, P=0.006) were independently associated with disease-free survival. The C-index of the scoring model (model 1) based on age, T stage, N stage, and dichotomous variables of the lymph nodes examined (<12 and ≥12) was 0.723, and the C-index of the scoring model (model 2) based on age, T stage, N stage, and multi-categorical variables of the lymph nodes examined (<12, 12 to <24, 24 to <36, and ≥36) was 0.726. A scoring system was established based on age, T stage, N stage, and multi-categorical variables of lymph nodes examined, the 3-year DFS of the low-risk (≤1), middle-risk (2 to 4) and high-risk (≥5) group were 96.3% (n=711), 89.0% (n=626) and 71.4% (n=313), respectively. Statistically significant difference was observed among groups (P<0.01). Conclusions: The number of lymph nodes examined was an independent prognostic factor for disease-free survival after curative surgery in patients with stage Ⅰ to Ⅲ colon cancer. Incorporating the number of lymph nodes examined as a multi-categorical variable into the T and N staging system could improve prognostic predictive validity.

[Analysis of prognosis and influencing factors of No. 253 lymph node metastasis in descending colon, sigmoid colon, and rectal cancer: a multicenter study].

Objectives: To analyze the influencing factors of No. 253 lymph node metastasis in descending colon cancer, sigmoid colon cancer, and rectal cancer, and to investigate the prognosis of No. 253 lymph node-positive patients by propensity score matching analysis. Methods: A retrospective analysis was performed on clinical data from patients with descending colon cancer, sigmoid colon cancer, rectosigmoid junction cancer, and rectal cancer who underwent surgery between January 2015 and December 2019 from the Cancer Hospital of the Chinese Academy of Medical Sciences, China-Japan Friendship Hospital, Peking Union Medical College Hospital, General Hospital of the Chinese People's Liberation Army, and Peking University Cancer Hospital. A total of 3 016 patients were included according to inclusion and exclusion criteria, comprising 1 848 males and 1 168 females, with 1 675 patients aged≥60 years and 1 341 patients aged<60 years. Clinical and pathological factors from single center data were subjected to univariate analysis to determine influencing factors of No. 253 lymph node metastasis, using a binary Logistic regression model. Based on the results of the multivariate analysis, a nomogram was constructed. External validation was performed using data from other multicenter sources, evaluating the effectiveness through the area under the receiver operating characteristic curve and the calibration curve. Using data from a single center, the No. 253 lymph node-positive group was matched with the negative group in a 1∶2 ratio (caliper value=0.05). Survival analysis was performed using the Kaplan-Meier method and Log-rank test. The Cox proportional hazards model was used to determine independent prognostic factors. Results: (1) The tumor diameter≥5 cm (OR=4.496,95%CI:1.344 to 15.035, P=0.015) T stage (T4 vs. T1: OR=11.284, 95%CI:7.122 to 15.646, P<0.01), N stage (N2 vs. N0: OR=60.554, 95%CI:7.813 to 469.055, P=0.043), tumor differentiation (moderate vs. well differentiated: OR=1.044, 95%CI:1.009 to 1.203, P=0.044; poor vs. well differentiated: OR=1.013, 95%CI:1.002 to 1.081, P=0.013), tumor location (sigmoid colon vs. descending colon: OR=9.307, 95%CI:2.236 to 38.740, P=0.002), pathological type (mucinous adenocarcinoma vs. adenocarcinoma: OR=79.923, 95%CI:15.113 to 422.654, P<0.01; signet ring cell carcinoma vs. adenocarcinoma: OR=27.309, 95%CI:4.191 to 177.944, P<0.01), and positive vascular invasion (OR=3.490, 95%CI:1.033 to 11.793, P=0.044) were independent influencing factors of No. 253 lymph node metastasis. (2) The area under the curve of the nomogram prediction model was 0.912 (95%CI: 0.869 to 0.955) for the training set and 0.921 (95%CI: 0.903 to 0.937) for the external validation set. The calibration curve demonstrated good consistency between the predicted outcomes and the actual observations. (3) After propensity score matching, the No. 253 lymph node-negative group did not reach the median overall survival time, while the positive group had a median overall survival of 20 months. The 1-, 3- and 5-year overall survival rates were 83.9%, 61.3% and 51.6% in the negative group, and 63.2%, 36.8% and 15.8% in the positive group, respectively. Multivariate Cox analysis revealed that the T4 stage (HR=3.067, 95%CI: 2.357 to 3.990, P<0.01), the N2 stage (HR=1.221, 95%CI: 0.979 to 1.523, P=0.043), and No. 253 lymph node positivity (HR=2.902, 95%CI:1.987 to 4.237, P<0.01) were independent adverse prognostic factors. Conclusions: Tumor diameter ≥5 cm, T4 stage, N2 stage, tumor location in the sigmoid colon, adverse pathological type, poor differentiation, and vascular invasion are influencing factors of No. 253 lymph node metastasis. No. 253 lymph node positivity indicates a poorer prognosis. Therefore, strict dissection for No. 253 lymph node should be performed for colorectal cancer patients with these high-risk factors.

Supplementation with N-carbamoylglutamate during the transition period improves the function of neutrophils and reduces inflammation and oxidative stress in dairy cows.

The aim of this study was to investigate the effects of N-carbamoylglutamate (NCG) supplementation during the transition period on the functions of blood polymorphonuclear neutrophils (PMN), inflammation, and oxidative stress in dairy cows. Thirty multiparous Chinese Holstein dairy cows at wk 4 before parturition were blocked into 2 groups by parity, body weight, and milk yield of previous lactation, and randomly allocated to 2 dietary treatments of basal diet supplemented without (control, n = 15) or with 20 g/d per cow of NCG (NCG, n = 15). The supplementation was carried out from d -21 to 21 relative to calving. Health incidents (mastitis, retained placenta, and lameness) were recorded, and blood samples were collected at d -21, -7, 0 (the calving date), 7, and 21 relative to parturition and analyzed for variables related to inflammation and oxidative stress. In addition, whole blood was collected at d 7 to isolate PMN and used for analysis of the expression of functional genes and from d -21 to 21 for determination of weekly hematological parameters. The number of lymphocytes was greater at d 7 in the blood of NCG cows. The plasma level of malondialdehyde was lower in the NCG group, and blood reactive oxygen species were lower at d 7, whereas total antioxidant capacity tended to be greater in the NCG group and glutathione peroxidase tended to be higher at d 21 in cows fed NCG, suggesting that NCG supplementation improved antioxidation in cows. In addition, the concentration of serum amyloid A was lower in NCG-fed animals during the postpartum stage. Blood concentrations of IL6 and tumor necrosis factor-α were lower and tended to be lower in NCG-fed animals at d 7, respectively. Meanwhile, the concentrations of IL6 tended to be lower in NCG-fed animals at d 21. Furthermore, the expression of S100A9 and MMP9 in the PMN was lower and tended to be lower, respectively, whereas the expression of ITGB2, XBP1 tended to be higher and expression of CLEC6A was higher in NCG-fed cows. Overall, our results indicated that supplementation with NCG during the transition period showed the beneficial effects on animal health, by improving PMN functions and alleviating inflammation status and oxidative stress in dairy cows.

[Comparison on efficacy between fascia-oriented versus vascular-oriented lateral lymph node dissection in patients with rectal cancer].

Objective: To compare the postoperative function, the short-term and long-term outcomes between fascia-oriented and vascular-oriented lateral lymph node dissection (LLND) in patients with rectal cancer. Methods: A retrospective cohort study was performed. Clinical data of patients who received total mesorectal excision (TME) with LLND at National Cancer Center, Cancer Hospital of Chinese Academy of Medical Science from January 2014 to December 2019 were retrospectively collected. Inclusion criteria were as follows: (1) rectal cancer was pathologically diagnosed, and the lower margin was below the peritoneal reflection. (2) resectable advanced rectal cancer with suspected lateral lymph node metastasis was evaluated based on rectal MRI assessment. (3) preoperative MRI showed lateral lymph node short diameter ≥5 mm and/or lymph node morphology (spike, blur, irregular) as well as heterogenous signal intensity. Lymph node shrinkage was less than 60% after receiving neoadjuvant therapy based on the reassessment of rectal MRI. (4) TME+LLND surgery was performed synchronously. Exclusion criteria were as follows: (1) previous history of pelvic surgery; (2) preoperative cystitis, urethritis, moderate and severe prostatic hyperplasia and other diseases resulting in abnormal urination function; (3) preoperative sexual dysfunction or loss of function; (4) patients receiving LLND due to lateral recurrence after TME; (5) distant metastasis of the tumor at initial diagnosis; (6) Incomplete collection of clinical data. A total of 73 consecutive patients were enrolled in this study. Based on the surgical approaches in performing LLND, patients were divided into fascia-oriented group (n=30) and vascular-oriented group (n=43). There were no significant differences in baseline data between the two groups (all P>0.05). The main outcome indicators of this study were the incidence of postoperative urinary and male sexual dysfunction, the efficacy, the number of lateral lymph nodes harvested and the detection rate of positive lymph nodes. Overall survival (OS) rates and progression free survival (PFS) rates were calculated by the Kaplan-Meier method and compared by log-rank test. Results: All patients in both groups completed surgery successfully. There were no significant differences in operation time, intraoperative blood loss, postoperative complications, and the length of hospital stay between the two groups (all P>0.05). In the whole group, the incidence of postoperative urinary dysfunction and male sexual dysfunction was 43.8% (32/73) and 62.5% (25/40), respectively. The median number of lateral lymph nodes harvested was 8.0(4.0,11.0) with a positive rate of 20.5%(15/73). Compared to the vascular-oriented group, the fascia-oriented group demonstrated a decreased rate of urinary dysfunction [26.7% (8/30) vs. 55.8% (24/43), χ(2)=6.098, P=0.014], lower rate of sexual dysfunction in males [6/15 vs. 76% (19/25), χ(2)=5.184, P=0.023], more harvested lateral lymph nodes [M (P25, P75): 9.5 (6.8, 15.3) vs. 6.0 (3.0, 9.0), Z=-2.849, P=0.004]. There was no significant difference in the positvie rate of lateral lymph nodes between the two groups [20% (6/30) versus 20.9% (9/43), χ(2)=0.009, P=0.923]. Three(4.1%) patients were lost during a median follow-up of 34 (1-66) months. The 3-year PFS and OS of the whole cohort were 69.5% and 88.3%, respectively. No significant difference in 3-year PFS rates (79.6% vs. 62.0%, P=0.172) and 3-year OS rates (91.2% vs. 85.9%, P=0.333) were observed between the fascia-oriented group and the vascular-oriented group (both P>0.05). Conclusion: Fascia-oriented LLND is associated with lower risk of postoperative urinary and male sexual dysfunction in patients with rectal carcinoma, and harvest of more lymph nodes, but no significant advantage in long-term survival.

Hypogalactia in mammary quarters adjacent to lipopolysaccharide-infused quarters is associated with transcriptional changes in immune genes.

Infusion of lipopolysaccharides (LPS) into a mammary gland can provoke inflammatory responses and impair lactation in both the infused gland and neighboring glands. To gain insight into the mechanisms controlling the spatiotemporal response to localized mastitis in lactating dairy cows, we performed RNA sequencing on mammary tissue from quarters infused with LPS, neighboring quarters in the same animals, and control quarters from untreated animals at 3 and 12 h postinfusion. Differences in gene expression were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Comparing mammary transcriptomes from all 3 treatments revealed 3,088 and 1,644 differentially expressed (DE) genes at 3 and 12 h, respectively. Of these genes, >95% were DE only in LPS-infused quarters and represented classical responses to LPS: inflammation, apoptosis, tissue remodeling, and altered cell signaling and metabolism. Although relatively few genes were DE in neighboring quarters (56 at 3 h; 74 at 12 h), these represented several common pathways. At 3 h, tumor necrosis factor (TNF), nuclear factor-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified by the upregulation of anti-inflammatory (NFKBIA, TNFAIP3) and cell adhesion molecule (VCAM1, ICAM1) genes in neighboring glands. Additionally, at 12 h, several genes linked to 1-carbon and serine metabolism were upregulated. Some responses were also regulated over time. The proinflammatory response in LPS-infused glands diminished between 3 and 12 h, indicating tight control over transcription to re-establish homeostasis. In contrast, 2 glucocorticoid-responsive genes, FKBP5 and ZBTB16, were among the top DE genes upregulated in neighboring quarters at both time points, indicating potential regulation by glucocorticoids. We conclude that a transient, systemic immune response was sufficient to disrupt lactation in neighboring glands. This response may be mediated directly by proinflammatory factors from the LPS-infused gland or indirectly by secondary factors released in response to systemic inflammatory signals.

Intramammary lipopolysaccharide infusion induces local and systemic effects on milk components in lactating bovine mammary glands.

Each quarter of the bovine mammary gland is an anatomically and functionally distinct gland. However, mastitis in one quarter may affect function of adjacent, uninfected glands. To investigate the mechanisms and potential mediators of these effects, we quantified early responses of the mammary gland to intramammary lipopolysaccharide (LPS) challenge, distinguishing between local and systemic effects. Ten multiparous cows over 70 d in milk were blocked into pairs by breed, cow-level somatic cell count (SCC), and milk yield. Within block, one cow was assigned to LPS treatment (T) such that both the front and the rear quarter of a randomly selected udder half received an infusion of 50 µg of LPS in 10 mL of saline (T-L); the contralateral quarters received only 10 mL of saline (T-S). Similarly, each paired control cow (C) received either 10 mL of saline (C-S) or no infusion (C-N) into udder halves. Cows were quarter milked twice daily, with foremilk samples (∼30 mL, front quarters) taken at -24, 0, 3, 6, 12, and 24 h relative to infusions. At 24 h, average milk yield in T-L and T-S quarters fell to 23 and 32% of pre-infusion levels, respectively. For T cows, systemic effects were observed by 3 h post-infusion as rectal temperature was elevated and foremilk fat concentration was reduced in both T-L and T-S. However, SCC and concentrations of l-lactate and total protein in foremilk indicated a local response to LPS: protein was transiently higher at 3 h, whereas SCC and lactate were higher at 6 h in T-L compared with T-S. Lactose concentration showed a local effect at 6 h, being lower in T-L than in T-S, and then a systemic effect at 12 h, being lower in both T-L and T-S than C quarters. Concomitant with changes in milk, systemic effects were also observed in blood. Plasma antioxidant potential and glucose concentration were lower in T cows than in C cows at 6 or 12 h, respectively, although neither variable remained different at 24 h. In summary, unilateral LPS infusion induced distinct, time-dependent effects on each milk component. Depending on the component, effects were local, systemic, or both, suggesting involvement of multiple different mediators that collectively result in systemic inhibition of milk production.

Protective Effects of Inorganic and Organic Selenium on Heat Stress in Bovine Mammary Epithelial Cells.

When dairy cows are exposed to high-temperature environment, their antioxidant capacity and productive performance decrease, leading to economic losses. Emerging evidence has shown that selenium (Se) can effectively alleviate heat stress in dairy cows; however, the cellular mechanism underlying this protection is not clear. The purpose of this study was to investigate and compare the protective effects of inorganic Se (sodium selenite, SS) and organic Se (selenite methionine, SM) in MAC-T (mammary alveolar cells-large T antigen, a bovine mammary epithelial cell (BMEC) line) cells during heat stress. MAC-T cells were treated in 4 ways unless otherwise described: (i) cells in the heat treatment (HT) group were cultured at 42.5°C for 1 h and then recovered in 37°C for another 12 h; (ii) the SM group was pretreated with organic Se for 2 h, cultured at 42.5°C for 1 h, and then recovered in 37°C for 12 h; (iii) the SS group was treated similarly to the SM group except that the cells were pretreated with inorganic Se instead of organic Se; and (iv) the control group was continuously cultured in 37°C and received no Se treatment. The results showed that heat shock at 42.5°C for 1 h triggered heat shock response, sabotaged the redox balance, and reduced cell viability in MAC-T cells; and pretreatment of cells with SM or SS effectively alleviated the negative effects of heat shock on the cells. However, the cells were much more sensitive to SS treatment but more tolerant to SM. In addition, two forms of Se appeared to affect the expression of different genes, including nuclear factor erythroid 2-related factor 2 (Nrf2) and inducible nitric oxide synthase (iNOS) in the SM group and thioredoxin reductase 1 (TXNRD1) in the SS group in Nrf2-ARE (antioxidant response element) antioxidant pathway and inflammation response. In summary, results showed the mechanistic differences in the protective effects of organic and inorganic Se on heat stress in BMECs.

An insufficient glucose supply causes reduced lactose synthesis in lactating dairy cows fed rice straw instead of alfalfa hay.

The objective of the present study was to investigate the nutrient availability for milk production in the mammary gland of lactating cows fed different forage-based diets. The 3 diets contained 30% corn stover (CS), 30% rice straw (RS), or 23% alfalfa hay plus 7% Chinese wild rye hay (AH) as a forage source. All diets contained 15% of DM as corn silage and 55% of DM as concentrate. The percentage of milk lactose was always lower in the RS-fed cows than in the cows fed AH or CS during the 12-wk feeding trial ( < 0.01). Ruminal propionate concentrations were lower in the RS group than in the AH group ( = 0.03). The ratio of insulin to glucagon in the mammary venous plasma was greater in the AH group than in the CS or RS group ( = 0.04). The abundance of the pyruvate carboxylase mRNA in the liver was lower in the RS group than in the AH or CS group ( = 0.04), and the abundance of mitochondrial phosphoenolpyruvate carboxykinase, IGF-1 receptor, and phosphofructokinase-liver, phosphofructokinase-muscle, and phosphofructokinase-platelet mRNA in the liver were lower in the RS group than in the AH group ( < 0.05). The mammary glucose uptake was greater in the AH-fed cows than in the CS- or RS-fed cows ( = 0.02). The mRNA abundance of the glucose transporters in the mammary gland was similar among the 3 treatments. The mRNA abundance of α-lactalbumin in the mammary gland of the cows fed RS tended to be greater compared with that of the cows fed AH or CS. The milk potassium concentration was greater in the cows fed RS than those fed AH or CS ( < 0.01). In summary, the insufficient ruminal propionate concentrations in the cows fed RS were associated with lower gluconeogenesis in the liver, resulting in the shortage of glucose supply for mammary utilization.

Nuclear factor-like factor 2-antioxidant response element signaling activation by tert-butylhydroquinone attenuates acute heat stress in bovine mammary epithelial cells.

Nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes. The Nrf2-ARE signaling plays a key role in the cellular antioxidant-defense system, but whether Nrf2 activation has protective effects against heat shock (HS) stress in mammary epithelial cells (MEC) remains unclear. The objective of this study was to determine whether tert-butylhydroquinone (tBHQ), a well-known Nrf2 activator, could attenuate heat stress-induced cell damage in MAC-T cells of the bovine MEC line. The MAC-T cells were exposed to HS (42.5°C for 1h) followed by recovery at 37°C to mimic HS. Compared with cells that were consistently cultured at normothermia (37°C), the cell viability levels significantly decreased after HS stress. In parallel, heat stress increased the reactive oxygen species levels and induced cellular apoptosis and endoplasmic reticulum stress. The MAC-T cells that were pretreated with tBHQ (10µM) for 2h followed by HS had a reduction in the loss of cell viability. The tBHQ pretreatment significantly decreased cellular reactive oxygen species levels and stress-related marker gene expression. The tBHQ-treated MAC-T cells showed strong Nrf2-ARE signaling activation and a nuclear accumulation of Nrf2 and upregulated expression of Nrf2-ARE downstream genes. Small interfering RNA silencing of Nrf2 in HS-treated MAC-T cells almost completely abolished the cytoprotective effects by tBHQ. Overall, our results demonstrated that HS could cause cell damage in cultured bovine MEC, and that activation of Nrf2 by tBHQ could attenuate HS-induced cell damage.

Facile one-pot synthesis of multi-yolk-shell Bi@C nanostructures by the nanoscale Kirkendall effect.

Multi-yolk-shell Bi@C nanostructures were prepared via a facile one-pot template-free hydrothermal approach. The prepared Bi@C nanostructures can act as a solid catalyst in the thermal decomposition of cyclotrimethylenetrinitramine (RDX) and display excellent catalytic activity, which highlights their application in the field of energetic materials.

Short communication: Protein kinase C regulates glucose uptake and mRNA expression of glucose transporter (GLUT) 1 and GLUT8 in lactating bovine mammary epithelial cells.

The aim of this study was to determine the role of protein kinase C (PKC) in regulating glucose uptake in lactating bovine mammary epithelial cells (BMEC). The BMEC were cultured and treated with different concentrations of phorbol 12-myristate 13-acetate (PMA;0, 10, 25, 50, 100, and 200 ng/mL), the classic activator of PKC, for 48 h. Compared with the cells with no PMA treatment, 50 and 100 ng of PMA/mL significantly stimulated the glucose uptake of the BMEC, whereas the glucose uptake by the cells treated with the lowest and the highest amounts of PMA (25 and 200 ng/mL, respectively) did not show a significant difference. Consistently, the mRNA expression of glucose transporter (GLUT) 1 and 8 showed a similar pattern of increase under the treatments of PMA. Furthermore, when the cells were pretreated with GF1090203X (0, 0.25, 0.5, 1, and 2 µM), an inhibitor of PKC, for 30 min before exposed to PMA (50 ng/mL), the PMA-induced glucose uptake and GLUT1 and GLUT8 expression were decreased by GF1090203X in a dose-dependent manner. These results demonstrate that PKC is involved in the regulation of glucose uptake by BMEC, and this function may work, at least partly, through upregulating the expression of GLUT1 and GLUT8.

Insulin stimulates glucose uptake via a phosphatidylinositide 3-kinase-linked signaling pathway in bovine mammary epithelial cells.

The aim of this study was to investigate the effects of insulin on glucose uptake in lactating bovine mammary epithelial cells (BMEC). Primary BMEC were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 and treated with different levels of insulin (0, 5, 50, and 500 ng/mL) for 48 h after a 24-h starvation without fetal calf serum. Compared with the control cells (0 ng of insulin/mL), cell proliferation was enhanced by insulin treatment at all tested levels. Insulin significantly increased glucose uptake at a concentration of 500 ng/mL. In addition, the protein synthesis inhibitor cycloheximide (0.5mg/mL) counteracted the insulin-elevated glucose uptake, thereby suggesting that newly synthesized transporter protein might take part in the insulin-induced glucose uptake. Furthermore, pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, did not influence the insulin-induced glucose uptake, but LY294002, a specific inhibitor of phosphatidylinositide 3-kinase, significantly reduced the insulin-stimulated glucose uptake. These results indicated that insulin-induced glucose uptake in BMEC may involve the phosphatidylinositide 3-kinase- but not mitogen-activated protein kinase-mediated signaling pathways.

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland.

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17ß-estradiol (InsHPrlE). The relative expression of ß-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of ß-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.

Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes.

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (K(m)) of 9.8 ± 3.0mM for 2-deoxy-d-glucose, similar to 11.7 ± 3.7 mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.

Osteopontin levels are elevated in patients with asthma.

Osteopontin (OPN) plays a role in T cell-mediated immunity, but its involvement in Th2-associated allergic responses, especially asthma, has not been investigated. This cross-sectional case-control study was designed to determine whether serum OPN levels are elevated in asthma patients and whether these correlated with disease severity. Serum samples were obtained from patients with mild (n = 42), moderate (n = 48) and severe (n = 39) asthma, and 41 healthy control subjects. OPN protein concentrations were measured by enzyme-linked immuno sorbent assay and were found to be significantly higher in all three asthma patient groups compared with healthy controls. There was no significant association between OPN concentration and disease severity. The data suggest a role for OPN in the pathogenesis of asthma. Further studies are needed to clarify the involvement of OPN in various aspects of asthma.

Expression and regulation of glucose transporters in the bovine mammary gland.

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.

Cloning and expression of bovine sodium/glucose cotransporter SGLT2.

The second member of the Na(+)/glucose cotransporter family, SGLT2, is a low-affinity active glucose transporter. In humans, it is predominantly located on the apical membrane of the S1 and S2 segments of renal proximal convoluted tubules, and, thus, may be mainly responsible for the reabsorption of D-glucose from the glomerular filtrate. By BLAST searching the GenBank database, we found expressed sequence tag sequences of SGLT2 in the cDNA library of bovine mammary tissues, indicating its expression in bovine mammary gland. To facilitate study of the mechanism of glucose reabsorption in bovine kidneys in maintenance of glucose homeostasis of lactating cows and the potential role of SGLT2 in the mammary gland, we cloned bovine SGLT2 and examined the distribution of its mRNA expression in bovine tissues. The full length mRNA of bSGLT2 is 2275 bp, and is predicted to encode a protein of 673 amino acids, with a molecular weight of approximately 73 kDa. The deduced amino acid sequence of bovine SGLT2 is 91, 90, 91, and 90% identical to human, rabbit, mouse, and rat SGLT2, respectively, and is 58 and 48% identical to bovine SGLT1 and SGLT5, respectively. The sequence of bSGLT2 contains several characteristically conserved sodium:solute symporter family signatures. Analysis of current bovine genomic data indicates that the bovine SGLT2 gene may consist of 14 exons. The major in vitro transcription and translation product of bovine SGLT2 cDNA migrated at an apparent molecular weight of 55 kDa. The SGLT2 mRNA was detected predominantly in bovine kidney as a 2.3-kb transcript, and at lower levels in all other bovine tissues examined, including the mammary gland, liver, lung, spleen, intestine, and skeletal muscle, as a 3.0-kb transcript. Expression of SGLT2 mRNA in bovine mammary gland increased more than 10-fold from late pregnancy to early lactation, similar to SGLT1. This indicates that SGLT2 may play a role in milk synthesis in the lactating mammary gland.

Cloning and expression of bovine sodium/glucose cotransporters.

The Na+-dependent glucose cotransporters (SGLT) are a family of glucose transporters that mediate an active, sodium-linked transport process against an electrochemical gradient. The SGLT are known to play important roles in absorption of dietary D-glucose and D-galactose from the intestinal lumen and in the reabsorption of D-glucose from the glomerular filtrate in kidney. To study the role and regulation of SGLT in tissues of lactating cows, we cloned and sequenced the full-length cDNA of bovine SGLT1 and SGLT5. Open reading frame analysis predicted that bovine SGLT1 is composed of 664 amino acids with a molecular weight of approximately 73 kDa, and SGLT5 is composed of 597 amino acids with a molecular weight of approximately 65 kDa. The deduced amino acid sequence of bovine SGLT1 is 48% identical and 66% conserved relative to that of bSGLT5. The amino acid sequence of bovine SGLT1 is 97, 88, 87, 86, 85, and 84% identical to sheep, mouse, rat, horse, human, and rabbit SGLT1, respectively. In contrast, the amino acid sequence of bSGLT5 is relatively divergent among species, being 85, 64, and 48% identical to rabbit, human, and rat SGLT5, respectively. Bovine SGLT retain the characteristic structural features of SGLT1 proteins described in other species, including membrane-spanning helices and glucose transporter motifs. The major in vitro transcription and translation product of bovine SGLT1 cDNA migrated at an apparent molecular weight of 52 kDa. In the presence of canine microsomal membranes, the translation product increased to 53 kDa, suggesting glycosylation. The SGLT1 mRNA was most abundant in bovine intestine, at intermediate levels in bovine kidney, and at lower levels in bovine mammary gland, liver, and lung. No SGLT1 mRNA was detected in bovine spleen, skeletal muscle, or testes. Expression of SGLT5 mRNA was found predominantly in bovine kidney, only at very low levels in bovine testes, skeletal muscle, and spleen, and was essentially undetectable in bovine mammary gland, liver, lung, and small intestine. Abundance of SGLT1 mRNA in bovine mammary gland increased more than 4-fold during late pregnancy and early lactation. The sequence and expression data reported in this paper lay the groundwork for future studies aimed at unraveling the functional roles of SGLT in supporting milk production and maintaining glucose homeostasis during lactation.

Purification of native myosin filaments from muscle.

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.

Glucose transporter gene expression in bovine mammary gland.

Transport of glucose across the plasma membrane of mammary epithelial cells is believed to be a passive process of facilitated diffusion mediated by facilitative glucose transporter(s). This article presents three lines of evidence that indicate the expression of sodium/glucose cotransporter (SGLT1) in the mammary gland of lactating and nonlactating cows. First, transcripts of SGLT1 mRNA ranging in size from 1.5 to 5.2 kb were detected in polyadenylated RNA preparations of mammary glands of lactating and nonlactating cows. Second, SGLT1 cotransporter protein was also detected in plasma membrane preparations of mammary glands of lactating cows. Third, partial amino acid sequence deduced from the reverse transcriptase-PCR fragment of SGLT1 from bovine mammary glands was similar to the sequence reported for ovine SGLT1. We conclude that mammary gland expression of SGLT1 mRNA and protein suggests that an active glucose transport system may be involved in glucose transport and metabolism in the mammary gland of dairy cows. However, the physiological significance of the expression of SGLT1 in mammary gland remains unknown.