Changes in tissue abundance and activity of enzymes related to branched-chain amino acid catabolism in dairy cows during early lactation.

Branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes the irreversible oxidative decarboxylation of branched-chain α-keto acids. This reaction is considered as the rate-limiting step in the overall branched-chain amino acid (BCAA) catabolic pathway in mammals. For characterizing the potential enzymatic involvement of liver, skeletal muscle, adipose tissue (AT), and mammary gland (MG) in BCAA metabolism during early lactation, tissue and blood samples were examined on d 1, 42, and 105 after parturition from 25 primiparous Holstein cows. Serum BCAA profiles were analyzed and the mRNA and protein abundance as well as the activity in the different tissues were assessed for the BCAA catabolic enzymes, partly for the branched-chain aminotransferase and completely for BCKDH. Total BCAA concentration in serum was lowest on d 1 after parturition and increased thereafter to a steady level for the duration of the experiment. Pronounced differences between the tissues were observed at all molecular levels. The mRNA abundance of the mitochondrial isoform of branched-chain aminotransferase (BCATm) was greatest in AT as compared with the other tissues studied, indicating that AT might be an important contributor in the initiation of BCAA catabolism in dairy cows. From the different subunits of the BCKDH E1 component, only the mRNA for the ß polypeptide (BCKDHB), not for the α polypeptide (BCKDHA), was elevated in liver. The BCKDHA mRNA abundance was similar across all tissues except muscle, which tended to lower values. Highest BCKDHA protein abundance was observed in both liver and MG, whereas BCKDHB protein was detectable in these tissues but could not be quantified. Adipose tissue and muscle only displayed abundance of the α subunit, with muscle having the lowest BCKDHA protein of all tissues. We found similarities in protein abundance for both BCKDH E1 subunits in liver and MG; however, the corresponding overall BCKDH enzyme activity was 7-fold greater in liver compared with MG, allowing for hepatic oxidation of BCAA transamination products. Reduced BCKDH activity in MG associated with no measurable activity in AT and muscle may favor sparing of BCAA for the synthesis of the different milk components, including nonessential AA. Deviating from previously published data on BCAA net fluxes and isotopic tracer studies in ruminants, our observed results might in part be due to complex counter-regulatory mechanisms during early lactation.

Detection of integrins in human cataract lens epithelial cells and two mammalian lens epithelial cell lines.

AIM: To compare the incidence of various integrin subunits in human cataract anterior lens epithelial cells (A-LEC) and in two mammalian LEC lines. METHODS: Circular sections of anterior capsules with attached LEC were obtained during cataract surgery. Integrin subunits were immunolocalised in these anterior LEC and in a human and rabbit LEC line, using four monoclonal antibodies specific for subunits alpha2, alpha3, and alpha5, and beta subunit 2. RESULTS: All of these subunits were found in at least a proportion A-LEC samples as follows: alpha2 71%, alpha3 92%, alpha5 62%, and beta2 24%. The human LEC line was immunoreactive for alpha2 and alpha3 only. The rabbit lens epithelial cell line was immunoreactive for alpha5 but there was no staining for alpha2, alpha3, or beta2. CONCLUSION: The A-LEC and mammalian LEC lines showed a similarity in their pattern of integrin expression. As these integrins are receptors for extracellular matrix (ECM) components, they are likely to be associated with the attachment and migration of LECs that precedes capsular opacification. Therefore these cell lines may be useful in the elucidation of mechanisms involved the pathogenesis of capsule opacification.

Patient satisfaction in our initial experience with endonasal endoscopic non-laser dacryocystorhinostomy.

BACKGROUND/AIMS: Endonasal endoscopic dacryocystorhinostomy (EN-DCR) is now a well-established procedure to relieve nasolacrimal duct obstruction. In the past, attempts have been made to comment on the anatomical success of the procedure. However, no studies have been conducted to evaluate patient satisfaction with the EN non-laser DCR procedure in comparison with the surgeon's experience. METHODS: Records of patients undergoing EN-DCR and external DCR (ET-DCR) surgery were reviewed. A telephone questionnaire was used to assess patient satisfaction with both procedures. Data were analyzed with Fisher's exact test and the chi-square test. RESULTS: Twenty primary EN-DCR's and 16 revision EN-DCR's were performed by the same surgeon (RM) over a three-year period. At last review, 89% of ET-DCR and 75% of EN-DCR procedures were noted to have a patent sac washout performed in the eye clinic. A telephonic interview revealed no significant difference between the surgical outcome [15/20 (75%)] and patient satisfaction [14/20 (70%)] with the primary EN-DCR procedure. Patient satisfaction with revision EN-DCR [10/16 (63%)] was slightly poorer than the surgical outcome recorded for revision EN-DCR [12/16 (75%)] but this was also not statistically significant. Telephonic interview was possible for 42/64 (66%) patients undergoing primary external ET-DCR's and a total of 36/42 (86%) patients were satisfied with the procedure. Patient age, laterality, duration of symptoms, previous ocular procedures or preexisting ocular disease and associated ENT procedures did not alter the surgical result or patient satisfaction in either group. CONCLUSIONS: This study suggests that patient satisfaction with endoscopic endonasal non-laser DCR for primary or revision DCR surgery is comparable to that with the external-DCR technique since there was no significant difference in patient satisfaction between the two groups of patients. Patient perception of their symptomatic improvement was lower (though not statistically significant) in relation to the final clinical assessment of the outcome of both primary and revision EN-DCR.

Expression of CD18, CD49b, CD49c and CD49e on lens anterior capsules in human cataracts.

PURPOSE: To examine the lens epithelial cells obtained from the anterior lens capsules removed during cataract surgery and detect various subclasses of the cell surface adhesion molecules known as integrins. METHODS: The circular sections of anterior capsules with attached lens epithelial cells (LECs) were obtained during cataract surgery from 28 patients. The lens capsules were immunohistochemically stained. RESULTS: CD49b CD49c, CD49e, and CD18 were detected in varying degrees in specimens obtained from human cataractous lenses. The positive percentages were 33, 75, 33, and 20%, respectively. The most striking feature was the increasing staining profiles towards the edges of the capsules (away from the central part of the lens capsules) for CD49c, suggesting that the LECs showed higher immunoreactivity for this antibody. The immunoreactivity for CD49b and CD49e was weaker. This was absent for CD18 in the central part of the lens capsules. CONCLUSION: The positive expression of antibodies suggests that specific integrin subunits were expressed in LECs of human cataracts. These results suggest that lens epithelial cells expressing CD49b, CD49c, CD49e, and CD18 might be precursors in the process of anterior lens epithelial cell (A cell) adhesion, and hence play a role in anterior capsule opacification or in subsequent migration and a possible role in posterior capsule opacification.

Pre-operative estimation of primary breast cancer size: a comparison of clinical assessment, mammography and ultrasound.

This prospective study aimed to determine which is the most precise modality for the pre-operative measurement of primary breast cancers: clinical palpation; mammography; or ultrasound. Analysis of the difference between the measurement of the maximum tumour diameter by these three modalities and by the histological measurement was performed in 210 cases. Clinical palpation tended to overestimate tumour size and gave the largest standard deviation of the difference. Ultrasound and mammography both gave a similar standard deviation of the difference, with ultrasound tending to underestimate tumour size. For all modalities, the standard deviation and the 95% confidence intervals of the difference increased with increasing tumour size. There is little difference between the precision of ultrasound and mammography in measuring tumour size. The wide 95% confidence intervals for any method of pre-operative tumour measurement should be considered when planning patient management.

Cytokeratin expression in breast cancer: phenotypic changes associated with disease progression.

Transition from a normal to a cancerous state is marked by alterations in the cytoskeletal structure of those cells involved. We have examined such changes to determine if these transitions are markers of disease progression. Cytokeratin (CK) protein and messenger RNA (mRNA) expression were examined in malignant and benign breast tissues. Flow cytometric results demonstrated a significant correlation between cytokeratin protein expression detected by 5D3 antibody, specific for cytokeratins 8, 18, and 19 and axillary node metastasis (P = 0.01). A threshold of positivity of 338,000 molecules/cell was determined and reflected the wide range in cytokeratin levels expressed by normal or benign tissues. Examination of cytokeratins 8, 18, and 19 revealed a consistent pattern of expression with respect to tumor grade. Only cytokeratin 19 showed significant correlation with increasing tumor size (P = 0.006). mRNA expression for cytokeratin 8 was significantly higher in node-positive compared with node-negative disease (P = 0.02). Cytokeratin 18 mRNA levels were significantly lower in both node-negative (P = 0.03) and node-positive (P = 0.02) patients when compared with benign samples. Increased levels of cytokeratin 18 mRNA showed an inverse relationship with protein expression (P = 0.05). The results indicate that cytokeratin expression in breast cancer may be associated with tumor progression. Furthermore, the alteration in the expression of individual cytokeratins deserves further investigation to determine the consequences of these changes with respect to cellular function.

The effect of 3-week tamoxifen treatment on oestrogen receptor levels in primary breast tumours: a flow cytometric study.

The effect of 3-week, preoperative tamoxifen treatment on oestrogen receptor (ER) levels, expressed by primary breast tumours, was examined. Patients (age-matched) with breast cancer, confirmed by fine-needle aspiration, were either treated with 20 mg ml(-1) oral tamoxifen per day or received no medication in the 3-week interval between assessment and surgery. Quantification of ER using flow cytometry was performed on the surgically removed tumour samples from tamoxifen-treated (n = 40) and control (n = 38, untreated) patient groups. The tumours were mechanically disaggregated, and saponin treatment rendered these cells permeable to antibodies. Using dual-parameter labelling with a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18/19 and a biotinylated antibody (DAKO-ER 1D5) directed against the oestrogen receptor, ER quantification was determined on a number of receptors per cell basis. Using QC quantum bead standards, ER levels in the epithelial cell population, the non-epithelial cell population and the whole-cell population (ER+) were calculated. ER levels were significantly lower in the total cell population than tamoxifen-treated patients (P = 0.002) when compared with the control (untreated) group. By using a gating procedure using 5D3 antibody positivity, a significantly lower level was detected on examining the cytokeratin-positive population alone (P = 0.006). Using a complementary gating technique, ER levels were quantified in the cytokeratin-negative cell population. Examination of this group of cells showed no significant difference between the levels of oestrogen receptor found in the tamoxifen-treated and untreated groups (P = 0.4). We have demonstrated that ER levels can be monitored by flow cytometry. ER levels in patients treated with tamoxifen 3 weeks before operation are significantly lower than in a comparative group of patients who received no drug. Furthermore, the most significant difference in receptor levels is seen by quantification of total ER levels expressed by all the tissue.