Flash labelling of S-phase cells in short-term organ culture of normal and pathological human endometrium using bromodeoxyuridine and tritiated thymidine

Normal hyperplastic and malignant endometrial specimens were labelled in vitro with bromodeoxyuridine (BrdU). S-phase cells were stained after DNA denaturation, using a monoclonal antibody to BrdU and an indirect immunoperoxidase method. Various conditions for BrdU uptake, DNA denaturation, and staining were tested. BrdU labelling was compared with autoradiography using tritiated thymidine, with good correlation. Glandular labelling indices of proliferative endometrium were significantly higher than both secretory and hyperplastic endometrium but were similar to carcinoma. Stromal labelling showed the same trend but the differences were not statistically significant.     

Lactate kinetics during passive and partially active recovery in endurance and sprint athletes

We investigated the effects of passive and partially active recovery on lactate removal after exhausting cycle ergometer exercise in endurance and sprint athletes. A group of 14 men, 7 endurance-trained (ET) and 7 sprint-trained (ST), performed two maximal incremental exercise tests followed by either passive recovery (20 min seated on cycle ergometer followed by 40 min more of seated rest) or partially active recovery [20 min of pedalling at 40% maximal oxygen uptake ( O_2max) followed by 40 min of seated rest]. Venous blood samples were drawn at 5 min and 1 min prior to exercise, at the end of exercise, and during recovery at 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 40, 50, 60 min post-exercise. The time course of changes in lactate concentration during the recovery phases were fitted by a bi-exponential time function to assess the velocity constant of the slowly decreasing component (₂) expressing the rate of blood lactate removal. The results showed that at the end of maximal exercise and during the 1st min of recovery, ET showed higher blood lactate concentrations than ST. Furthermore, ET reached significantly higher maximal exercise intensities [5.1 (SEM 0.5) W · kg^–1 vs 4.0 (SEM 0.3) W · kg^–1,P < 0.05] and O_2max [68.4 (SEM 1.1) ml · kg^–1 · min^–1 vs 55.5 (SEM 5.1) ml · kg^–1 · min^–1,P < 0.01]. There was no significant difference between the two groups during passive recovery for ₂ During partially active recovery, ₂ was higher than during passive recovery for both groups (P < 0.001), but ET recovered faster and sooner than ST (P < 0.05). Compared to passive recovery, the ₂ measured during partially active recovery was increased threefold in ET and only 1.5-fold in ST. We concluded that partially active recovery potentiates the enhanced ability to remove blood lactate induced by endurance training.     

The influence of inspired oxygen on the oxygen uptake response to ramp exercise

The relation between and work rate (WR) was examined in seven male subjects who performed ramp (1 W·3 s^–1) two-legged cycle ergometry to exhaustion while inspiring either hypoxic (12% O₂), normoxic (21% O₂), or hyperoxic (40% O₂) air. The anaerobic threshold was estimated from respiratory gas exchange data and is thus referred to as the respiratory gas exchange threshold (RGET). Prior to the RGET, the was greater under normoxic [mean (SD); 10. 19(1.04) ml O₂·min^–1·W^–1] and hyperoxic [10.44 (0.72)] conditions compared with hypoxia [9.34 (0.89)]. Above the RGET, the for hypoxia [8.91 (0.63)], normoxia [10.40 (0.77)], and hyperoxia [11.08 (0.48)] were all significantly different from each other. These data indicated that for two-legged, cycle, ramp ergometry in normoxia below the RGET, both the and response time was constant. Above the RGET, the normoxic response was the net result of a declining and a longer response time to the unsteady state character of a ramp exercise protocol.     

The VO2 response to exhaustive square wave exercise: influence of exercise intensity and mode

We investigated the oxygen uptake (O₂) response to exhaustive square wave exercise of approximately 2, 5 and 8 min duration in cycling and running. Nine males completed a ramp test and three square wave tests on a motorised treadmill and the same four tests on a cycle ergometer, throughout which gas exchange was assessed (Douglas bag method). The peak O₂ from the ramp test was higher for running than for cycling [mean (SD): 58.4 (2.8) vs. 55.9 (3.7) ml.kg^–1.min^–1; P=0.04]. However O_2max (defined as the highest O₂ achieved in any of the four tests) did not differ between running and cycling [60.0 (2.9) vs. 58.5 (3.3) ml.kg^–1.min^–1; P=0.15]. The peak O₂ was similar (P>0.1) for the 5 and 8 min square wave tests [98.5 (1.8) and 99.2 (2.3) %O_2max for running; 97.0 (4.2) and 97.5 (2.0) %O_2max for cycling] but lower (P<0.001) for the 2-min test [91.8 (2.5) and 89.9 (5.5) %O_2max for running and cycling respectively]. O₂ increased over the final two 30-s collection periods of the 2-min test for cycling [O₂=0.18 (0.15) l.min^–1; P<0.01] but not running [O₂=0.00 (0.09) l.min^–1; P=0.98]. We conclude that in the aerobically fit the peak O₂ for square wave running or cycling at an intensity severe enough to result in exhaustion in approximately 2 min is below O_2max. In running, O₂ plateaus at this sub-maximal rate.     

A Novel Macroinitiator for the Synthesis of Triblock Copolymers via Atom Transfer Radical Polymerization: Polystyrene‐block‐poly(bisphenol A carbonate)‐block‐polystyrene and Poly(methyl methacrylate)‐block‐poly(bisphenol A carbonate)‐block‐poly(methyl methacrylate)

Summary: Bis(hydroxy)telechelic bisphenol A polycarbonate (PC) was prepared via melt polycondensation of bisphenol A (BPA) and diphenyl carbonate (DPC) using lanthanum(III ) acetylacetonate as a catalyst for transesterification. Subsequently, the polycarbonate was converted to a bifunctional macroinitiator for atom transfer radical polymerization (ATRP) with the reagent, α‐chlorophenylacetyl chloride. The macroinitiator was used for the polymerization of styrene (S) and methyl methacrylate (MMA) to give PS‐block‐PC‐block‐PS and PMMA‐block‐PC‐block‐PMMA triblock copolymers. These block copolymers were characterized by NMR and GPC. When styrene and methyl methacrylate were used in large excess, significant shifts toward high molecular weights were observed with quantitative consumption of the macroinitiator. Several ligands were studied in combination with CuCl as the ATRP catalyst. Kinetic studies reveal the controlled nature of the polymerization reaction for all the ligands used.

Formation of a bifunctional ATRP macroinitiator by esterification of bis(hydroxy)telechelic PC with α‐chlorophenylacetyl chloride.     

Characteristics of the transport of oxalate and other ions across rabbit proximal colon

In order to characterize oxalate handling by the P2 segment of the rabbit proximal colon, the fluxes of [¹⁴C]oxalate, ²²Na⁺, and ³⁶Cl^– were measured in vitro using conventional short-circuiting techniques. In standard buffer the proximal colon exhibited net secretion of Na⁺ (–2.31±0.64 equiv cm^–2 h^–1), negligible net Cl^– transport, and net secretion of oxalate (–12.7±1.6 pmol cm^–2 h^–1). Replacement of buffer Na⁺ or Cl^– abolished net oxalate secretion, while HCO ₃ ^– -free media revealed a net absorption of oxalate (19.3±4.2 pmol cm^–2 h^–1) and stimulated NaCl absorption. Mucosal amiloride and dimethylamiloride (1 mM) significantly reduced the unidirectional fluxes of oxalate and enhanced sodium secretion by decreasing J _ms ^Na . The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 0.1 mM, both sides) reduced the unidirectional fluxes of oxalate and chloride. Serosal epinephrine (50 M) stimulated oxalate absorption (21.3±6.3 pmol cm^–2 h^–1) and sodium absorption (5.71±1.20 equiv cm^–2 h^–1), whereas dibutyryl-cAMP enhanced oxalate secretion (–43.4±6.9 pmol cm^–2 h^–1) and stimulated chloride secretion (–7.27 ±0.64 equiv cm^–2 h^–1). These results indicate that the P2 segment of the proximal colon possesses (a) secretory as well as absorptive capacities, (b) oxalate fluxes that are mediated by pathways involving Na⁺, Cl^–, HCO ₃ ^– transport and (c) a net oxalate flux that is sensitive to absorptive and secretory stimuli.     

O2 Uptake in hyperthyroidism during constant work rate and incremental exercise

Summary To investigate the effect of hyperthyroidism on the pattern and time course of O₂ uptake ( O₂) following the transition from rest to exercise, six patients and six healthy subjects performed cycle exercise at an average work rate (WR) of 18 and 20 W respectively. Cardiorespiratory variables were measured breath-by-breath. The patients also performed a progressively increasing WR test (1-min increments) to the limit of tolerance. Two patients repeated the studies when euthyroid. Resting and exercise steady-state (SS) O₂ (ml·kg^–1·min^–1) were higher in the patients than control (5.8, SD 0.9 vs 4.0, SD 0.3 and 12.1, SD 1.5 vs 10.2, SD 1.0 respectively). The increase in O₂ during the first 20 s exercise (phase I) was lower in the patients (mean 89 ml·min^–, SD 30) compared to the control (265 ml·min^–1, SD 90), while the difference in half time of the subsequent (phase 11) increase to the SS O₂ (patient 26 s, SD 8; controls 17 s, SD 8) were not significant (P = 0.06). The OZ cost per WR increment ( O₂/WR) in ml·min^–1·^–1, measured during the incremental period (mean 10.9; range 8.3–12.2), was always within two standard deviations of the normal value (10.3, SD 1). In the two patients who repeated the tests, both the increment of O₂ from rest to SS during constant WR exercise and the O₂/WRs during the progressive exercise were higher in the hyperthyroid state than during the euthyroid state. While both resting and exercise O₂ are increased in the hyperthyroid patients, the O₂ cost of a given increment of WR is within the normal range. However, a small reduction in the O₂ requirement to perform exercise following treatment of the hyperthyroid state suggests a subtle change O₂ cost of muscle work in this disease.     

The prognostic value of Ki67 immunostaining in non-Hodgkin's lymphoma

The monoclonal antibody Ki67 recognizes an antigen expressed in all phases of the cell cycle except Go. It has been used in 141 biopsies from 138 patients with non-Hodgkin's lymphoma to identify proliferating cells in histological sections. A Ki67 index (the number of Ki67 positive tumour cells divided by the sum of Ki67 positive and negative tumour cells) has been derived by counting 1000 cells in each case. A correction for the presence of non-tumour cells has been incorporated by counting non-tumour cells in serial sections stained with a panel of other antibodies. A very strong correlation between a low Ki67 index (less than 20 per cent) and low grade histology and a high Ki67 index (greater than 20 per cent) and high grade histology was found (Chi2 = 98.0). Ninety-one patients could be analysed for survival and those with low grade lymphoma (n = 38) who had a relatively high Ki67 index (greater than 5 per cent) had a worse survival than those with an index of less than 5 per cent (p less than 0.05). In contrast, there was a trend for those patients with high grade disease with a very high Ki67 index (greater than 80 per cent) to have a better survival than those with a lower index (less than 80 per cent). The patients with high grade disease who achieved complete remission or good partial remission and had a Ki67 index of less than 80 per cent were more likely to relapse than those with an index of greater than 80 per cent (p less than 0.04). These findings could not be explained by the effect of other prognostic factors such as age, stage, or serum albumin. While the use of Ki67 immunostaining has potential drawbacks, it appears to be a simple and reproducible method of determining a tumour proliferative index which provides relevant clinical data.     

Proliferation Kinetics of Rat Kupffer Cells after Partial Hepatectomy Immunohistochemical and UItrastructural Analysis

In an effort to settle the conflicting views on the proliferation kinetics of Kupffer cells (Kc), we performed 2/3 partial hepatectomy on rats injected with Pelikan ink. Using an anti-rat macrophage monoclonal antibody, ED 2, we evaluated the numerical changes in total, carbon-positive ED 2⁺ cells and carbon-negative ED 2⁺ cells in the portal and central area. We also analyzed the ultrastructure and peroxidase cytochemistry of various types of cells observed during regeneration. The total numbers of ED 2⁺ cells in the remaining liver increased rapidly from day 2 to 5, and the number of dividing ED 2⁺ cells reached a maximum on day 2. Thus, the numerical increase in ED 2⁺ cells corresponded to the division phase. In contrast, the carbon-labeling experiment showed a continuous increase of carbon negative ED 2⁺ cells from day 2 to 7. In the central area where division was less frequent, the proportion of carbon-positive cells decreased markedly to 50% on day 7, as against 97% in control rats. These findings suggest the possibility of an influx of carbon-negative Kc in addition to cell division. Ultrastructurally, the presence of carbon-negative "small Kc" and "immature Kc" with morphological features different from those of carbon-positive Kc was demonstrated. Such carbon-negative Kc with a high nucleus-to-cytoplasm ratio and rather few phagosomes, were not observed in control rats. Furthermore, we demonstrated two types of possible precursor cell, i.e. "transitional" forms between monocytes and Kc, and "immature macrophages". The former showed peroxidase activity in some lysosomes as well as in the rough endoplasmic reticulum and nuclear envelope. Our result indicated that the proliferation kinetics of Kc depend upon both local proliferation and influx.     

A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes

A new method for the measurement of phagocytosis of Candida albicans by human polymorphonuclear leucocytes (PMN) is described using a fluorescence activated cell sorter. We have used acridine orange to discriminate between PMN which have internalised yeast particles and those which have not. This method allows accurate measurement of particle phagocytosis as an event distinct from particle adherence. It also permits detailed examination of the kinetics of phagocytosis, the study of which is likely to be of value in the investigation of diseases where abnormalities of PMN function are suspected.