Comparative study of the pollen protein contents in two major varieties of Cupressus arizonica planted in Tehran

During past few years, the Cupressus arizonica has been abundantly planted in Tehran, causing a significant increase of allergic diseases from the middle of winter to the beginning of spring. The aim of this study was the comparison of pollen protein content in two major varieties of C. arizonica planted in Tehran, including C. arizonica var. arizonica and C. arizonica var. glabra, in order to determine pollen's specificity of each variety and also to find out whether environmental conditions can influence pollen protein contents and its allergenic components. Pollen grains were directly collected from mature male cones of trees planted in different areas of the city. Pollen's proteins were extracted, and were analyzed by SDS PAGE. Total protein content of pollen extracts was measured by Bradford assay. Our investigations revealed noticeable differences in protein content of each variety. Bradford protein assay showed a higher total protein content in C. arizonica var. arizonica pollen extracts. A new major protein, with an approximate molecular weight of about 35 kDa was detected in both varieties. Immunoblotting using the serum of a cypress allergic subject showed that the protein with 35 kDa was also the major allergen of both varieties in pollen extracts. These results showed that there are some intraspecie specificities in Arizona cypress pollens. The major allergen of Cupresuss arizonica pollen, Cup a 1 (45 kDa), has been reported as the most representative protein in pollen extracts of Mediterranean countries, but in our autochthon extracts of both varieties, a protein band at 35 kDa was more representative. These observations seem to indicate that C. arizonica pollen protein content may be influenced by environmental conditions. Moreover, Immunoblot results provided a reliable indication on the allergenic activity of this new major protein band at 35 kDa. The confirmation of these aspects would facilitate the preparation of an effective extract, improving the diagnosis of the allergy to the Cupressus arizonica pollen.     

In vitro and in vivo studies on Leydig cell function in old rats.

Young adult (3 months old) and old (26-28 months old) male Wistar rats were studied. Testicular weight was 1.66 g (range: 1.33--2.86) in the younger group and 1.62 g (range: 1.04--1.90) in the older group. The total Leydig cell volume as measured by a quantitative histometric method was significantly larger in the old animals (-x = 0.153 ml vs. -x = 0.089 ml). The testicular HCG binding capacity was 2.54 ng HCG per 100 mg tissue (range: 1.88--3.77) in the younger animals and 1.83 ng HCG per 100 mg tissue (range: 0.80--3.02) in the older ones (P less than 0.01). Plasma testosterone was on an average 242 ng/100 ml (range: 72--1162) in the young adult rats and 91 ng/100 ml (range: 23--277) in the older rats. Plasma LH was only slightly (P less than 0.05) decreased and was 49 ng LH-RP-1/ml (range: 14--120) in the younger group and 40 ng LH-RP-1 ng/ml (range: 5--98) in the older. When the testicular tissue pieces were incubated with different doses of HCG or dibutyryl cAMP and testosterone production was measured, identical dose-response curves for old and young tissue were obtained. After in vitro incubation with a NADPH generating system, which under the conditions used was a much stronger stimulus for steroid biosynthesis than HCG or dibutyryl cAMP, the tissue of young rats produced about twice the amount of testosterone precursors (pregnenolone, progesterone, 17alpha-hydroxyprogesterone and androstenedione) than old tissue.     

Correlation of in vitro and in vivo biological activities during the partial purification of thrombopoietin.

We have evaluated three in vitro assays for megakaryocyte maturation as monitors of biological activities of thrombopoietin (TPO). The in vitro measurements, that is, potentiation of murine megakaryocyte colonies in soft agar cultures, growth of single murine megakaryocytes in soft agar, and acetylcholinesterase production in liquid cultures of murine bone marrow cells, were correlated with measurements of thrombopoiesis stimulatory activity in vivo, based on labeling of newly formed platelets with [75Se]selenomethionine. Protein fractions produced during the purification of TPO from the plasma of thrombocytopenic rabbits were used to evaluate the in vitro assays. Potentiation of murine megakaryocyte colony growth in soft agar was least valuable as a TPO assay, due to variability. Growth of single megakaryocytes in vitro, in a serum-free, agar culture system, correlated well with measurement of thrombopoiesis-stimulating activity in vivo, in regard to increases in specific activity during the purification, and was the most sensitive of the three assays. The serum-free, liquid culture assay also correlated well with the in vivo assay, and it had the additional advantage of being feasible for evaluation of the large numbers of protein fractions produced by high-resolution chromatographic procedures. Using the liquid culture system to assay fractions from gel permeation high performance liquid chromatography, a biologically active fraction with a molecular weight range of 40-47 kd was identified.     

In vivo and in vitro comparisons of biological activities of bovine, ovine and rat CRF (corticotrophin-releasing factor)

The biological activity of partially purified bovine hypothalamic CRF (corticotrophin- releasing factor) was compared to those of synthetic CRFs (ovine, rat) sauvagine and vasopressin in vivo and in vitro. ACTH-primed hypophysectomized rats with heterotopically transplanted pituitaries and medial basal hypothalamic ablation (H-T + MBHA ), and intact rats pre-treated with chlorpromazine, morphine and Nembutal (C-M-N) were used for in vivo CRF assays. Perifused rat adenohypophyseal fragments were employed for in vitro studies. CRF-A (void volume fractions, 'big' CRF) and CRF-B (Kav = 0.583) purified from bovine hypophyseal stalk, synthetic ovine and rat CRF, and sauvagine all induced significant stimulation of ACTH and/or corticosterone secretion in these systems. Synthetic ovine and rat CRF and sauvagine showed comparable CRF potency. The CRF dose-response slopes for bovine CRF were somewhat steeper than those for ovine CRF or sauvagine in the in vitro system. Vasopressin had the least steep dose-response slope. Intravenous bolus administration of ovine CRF caused a more prolonged (greater than 20 min) elevation of plasma ACTH compared to a relatively short duration after bovine CRF-A. These data suggest that bovine hypothalamus contains substance(s) which exhibits different CRF characteristics from those of ovine CRF.     

In vitro and in vivo activities of sparfloxacin (AT-4140) against Mycobacterium tuberculosis.

The in vitro and in vivo activities of sparfloxacin (AT-4140) against M. tuberculosis are reported. The MICs of sparfloxacin for 50% and 90% of 18 clinical isolates were, respectively, 0.25 and 0.5 mg/l, one or two dilutions lower than that of ciprofloxacin and ofloxacin. In mice infected intravenously with 0.1 mg M. tuberculosis H37Rv strain, the minimal effective dosage of sparfloxacin, as assessed by survival rate, spleen enlargement and gross lung lesions, was 12.5 mg/kg. The activities of various regimens were in the following rank order: INH 25 mg/kg = sparfloxacin 50-100 mg/kg greater than ofloxacin 300 mg/kg greater than (or =) sparfloxacin 25 mg/kg greater than sparfloxacin 12.5 mg/kg greater than (or =) ofloxacin 200 mg/kg greater than ofloxacin 100 mg/kg greater than (or =) negative control. Therefore, on a weight to weight basis, sparfloxacin was six to eight-fold more active against M. tuberculosis infection in mice than ofloxacin. In addition, WIN 57273, a new broad-spectrum fluoroquinolone, at a dosage of 100 mg/kg daily, was inactive against M. tuberculosis infection.     

Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones

Gonadotropins are synthesized and released in different molecular forms. In this article, we present evidence that the glycosylation variants of human pituitary FSH exhibit differential and divergent effects at the target cell level and that less sialylated, short-lived variants may exert significant effects in in vivo conditions. Less acidic/sialylated glycoforms (elution pH value 6.60-4.60 as disclosed by high resolution chromatofocusing of anterior glycoprotein extracts), induced higher cAMP release, estrogen production and tissue-type plasminogen activator (tPA) enzyme activity as well as cytochrome P450 aromatase and tPA mRNA expression in cultured rat granulosa cells than the more acidic analogs (pH<4.76). By contrast, the more acidic/sialylated glycoforms induced higher alpha-inhibin subunit mRNA expression than their less acidic counterparts. In cumulus enclosed oocytes isolated from mice ovaries, addition of less acidic isoforms induced resumption of meiosis more efficiently than the more acidic analogs. Interestingly, the least acidic isoform (pH>7.10) behave as a strong antagonist of several FSH-mediated effects. Assessment of the in vivo effects of the isoforms on granulosa cell proliferation in follicles from immature rats, revealed that short-lived isoforms were equally or even more efficient than their more acidic counterparts in maintaining granulosa cell proliferation when administered immediately after hypophysectomy. These results show that the naturally occurring human FSH isoforms may exhibit differential or even unique effects at the target cell level and that factors other than the metabolic clearance rate of the molecule (including receptor-binding affinity and capability of the ligand to activate its receptor and trigger intracellular signaling) also play an important role in determining the net in vivo effects of a particular FSH variant.     

In vitro and in vivo intravascular ultrasound imaging.

This paper presents our experience with intravascular ultrasound imaging of animal and human arteries in vitro and in vivo using a high-frequency (20 M Hz) ultrasound transducer. In vitro, 32 human coronary artery segments were imaged with intravascular ultrasound and compared with corresponding histological sections. Ultrasound and histology measurements correlated significantly (P less than 0.0001) for coronary artery cross-sectional area (r = 0.94), lumen cross-sectional area (r = 0.85) and wall thickness (r = 0.92). In vivo, 19 sheep and eight human common femoral arteries were imaged and the angiographic lumen diameter of 14 animal and six human arteries was compared to the diameter of the corresponding ultrasound images. Significant correlations were found for lumen diameter in animals and humans (P less than 0.001, r = 0.91 and P less than 0.0001, r = 0.96, respectively). These studies demonstrate that this technique can provide high resolution images of arterial vessels and may have unique advantages in diagnosing atherosclerotic vascular disease and in catheter based therapies.     

In vitro and in vivo activities of ofloxacin against Mycobacterium leprae infection induced in mice

Ofloxacin, a new quinolone, exhibits bactericidal activity against Mycobacterium leprae in mice, both in vivo and in vitro.     

左金丸及其主要单体成分对大肠癌的干预作用

目的: 探讨左金丸及其主要单体成分在体内体外对大肠癌发生发展的干预作用.方法: 培养人大肠癌细胞株HT29, 选择处于对数生长期的细胞, 分别用不同浓度的盐酸小檗碱(26.25、52.5、105、210、420 μmol/L)、吴茱萸碱(5、10、15、20、25、30 μmol/L)处理HT29细胞24、48、72、96 h, 筛选最适作用浓度与时间, 在这一浓度与时间作用下,用活细胞计数法检测左金丸的主要单体成分小檗碱和吴茱萸碱对HT29细胞增殖的抑制作用, TRAP法检测HT29细胞端粒酶活性, 取HT29细胞, 用生理盐水制成单细胞悬液, 调整细胞浓度为5×105/0.2 mL, 注射于裸鼠左侧胁腹部皮下, 制作HT29细胞荷瘤裸鼠肿瘤模型. 随机将裸鼠分为4组: 对照组、左金丸组、AZT组和协同组, 观察左金丸和叠氮胸苷(AZT)对瘤体生长及端粒酶活性的影响. 利用致癌剂1, 2-二甲基酰肼(DMH, 25 mg/kg)诱导大鼠大肠癌模型, 同样随机将大鼠分为4组, 观察左金丸和AZT对大鼠大肠癌发生发展的干预作用.结果: 在离体实验中, 处理24-72 h, 105 μmol/L盐酸小檗碱和15 μmol/L吴茱萸碱对HT29细胞增殖的抑制作用呈成正向的线性量效关系, 吴茱萸碱(7.5、15、30 μmol/L)作用后的抑制率分别为39.3%±2.13%, 52.8%±5.34%和64.1%±7.19%, 盐酸小檗碱(52.5、105、210 μmol/L)作用后的抑制率分别为44.1%±3.97%, 55.9%±4.12%和65.3%±6.94%. 105μmo l/L小檗碱和15 μmo l/L吴茱萸碱处理HT29细胞72 h, 小檗碱和吴茱萸碱可以有效抑制HT29细胞端粒酶的活性. 在荷瘤裸鼠实验中, 左金丸和AZT对瘤体大小无明显影响,对端粒酶活性有微弱影响. 第11周, 对照组、左金丸组、AZT组和协同组大鼠肿瘤发生率分别为20%、0%, 10%和10%, 左金丸作用优于AZT, 但34 wk各组间无明显差异.结论: 离体实验中, 左金丸的主要成分可以有效抑制HT29细胞的增殖和端粒酶的活性;裸鼠移植瘤实验中, 观察不到左金丸的药效;1,2-DMH诱导大肠癌实验模型中, 左金丸可以有效阻碍早期癌症的发生和发展.     

In vivo and in vitro evidence of an adenosine-mediated mechanism of calcium entry blocker activities

Drugs such as dipyridamole (200 micrograms/kg/min), an adenosine uptake inhibitor, and theophylline (300 micrograms/kg/min), an adenosine receptor antagonist, respectively increased and decreased postischemic hyperemia in normal subjects, as well as in POAD patients. Moreover, dipyridamole pretreatment was able to antagonize the reduction of peak flow induced by nifedipine, and the potentiating effect of flunarizine on postischemic hyperemia was affected significantly by theophylline, thus suggesting a possible interference of calcium entry blocker drugs with the endogenous adenosine system. In a cellular model (polymorphonuclear leukocytes--PMN) the inhibitory effect of calcium entry blockers on stimulated functions (degranulation and free radical production) was highly antagonized by theophylline. Finally, a 1H-NMR spectroscopy study showed a binding interaction between adenosine and flunarizine on the cell membrane. An adenosine-receptor coupling to the calcium entry blocker channels is suggested.     

In vitro biological activities of the haemopoietic growth factors: implications for their clinical use.

The recent availability of several haemopoietic growth factors in purified recombinant form suitable for clinical use has prompted their use in haematology and oncology. The knowledge of their various effects in vitro on cell proliferation, differentiation and function and of their target cell populations will help in the design of treatment protocols with regard to selection of the best growth factor (or combination of them) for specific desired effects.     

In vitro and in vivo production of CFU-S-stimulating activities after 5-Gy total body irradiation

In vitro and in vivo production of CFU-S-stimulating activities have been described after 5-Gy irradiation of mice using a diffusion chamber-incubation technique. The experimental results showed that bone marrow obtained 15 min after 5 Gy total body irradiation did not present detectable CFU-S stimulating activity, but was able to produce such activity in vitro within 20 h. Once the activity had been produced, 2.5 h was enough to stimulate the proliferation of CFU-S. When CFU-S were incubated for 20 h in the peritoneal cavities of mice that had been irradiated with 5 Gy immediately prior to the insertion of the chambers, or five days earlier, a marked stimulation of CFU-S turnover was observed, demonstrating the in vivo production of some humoral activity capable of stimulating CFU-S.     

Mechanical activities of trachea as measured in vitro and in vivo.

The phenomenon of spontaneous contractions as measured in vivo and in vitro (isolated guinea pig trachea) is described. In vitro contractility is defined in terms of active tension (AT), the maximum rate of tension development (dT/dt) and time to peak tension (TTP). The length-tension relationship was established for spontaneous contractions of isolated guinea pig trachea. The effect of the temperature on spontaneous activities was also studied. An increase in temperature from 37.5 to 41.5C produced an increase in the maximum rate of tension development (dT/dt( and a decrease in the time to peak tension (TTP). A comparable decrease in temperature, from 37.5 to 33.5C produced slight but not statistically significa-nt increases in active tension, dT/dt and TTP. Frequency of the spontaneous contractions varied directly with changes in temperature. Employing a micro-strain-gauge transducer a rhytmic change of tracheal diameter has been observed in vivo in both guinea pigs and rabbits. Administration of atropine and isoproterenol in those experiments transiently abolished the appearance of these rhythmic changes of tracheal diameter.     

Heparin-antithrombin III binding. In vitro and in vivo studies

Heparin antithrombin III binding was studied by crossed immunoelectrophoresis. In plasma and purified antithrombin III standard, multicomponent patterns were obtained with low concentrations of mucosal heparin. There is evidence that antithrombin III may bind more than one heparin molecule. At high heparin concentration (greater than 16 U/ml), single symmetrical peaks were obtained. Serum samples showed two antithrombin III peaks due to a decreased heparin binding of the slower peak (2.1-3.9 times), which was probably antithrombin III-activated procoagulant complexes. Heparin analogue (A 73025) also bound antithrombin III in vitro but the mobility of the peak was slower than with mucosal heparin and only a single peak was obtained in serum samples. Radioimmunoassay showed a decreased binding of antithrombin III antibody to heparin-antithrombin III complex. Venous occlusion to the forearm resulted in a slow second peak in the plasma. Heparin therapy gave rise to a double peak in the plasma antithrombin III profile and with continuous infusion, quantitative decreases were noted in all subjects studied, two of whom rethrombosed at the end of 7 days therapy.