三种方法诱导熊本牡蛎三倍体的比较研究

为获得高效的熊本牡蛎的三倍体诱导方法, 分别比较了6-DMAP、高盐和低盐3种诱导方法在不同的诱导浓度(盐度)、起始诱导时间和持续诱导时间下的卵裂率、孵化率和三倍体率, 同时比较了3种方法获得的幼虫的生长、存活和三倍体率变化情况。结果表明, 在6-DMAP诱导组中, 三倍体率和诱导效率分别可达37.97%—58.01%和34.30%—42.50%, 培育期间幼虫的平均存活率27.19%, 生长率13.03 μm/d, 三倍体率降低了24.94%; 在低盐诱导组中, 三倍体率和诱导效率分别可达7.32%—42.25%和2.17%—31.41%, 培育期间幼虫的平均存活率33.92%, 生长率12.71 μm/d, 三倍体率降低了20.64%; 在高盐诱导组中, 三倍体率和诱导效率分别可达7.47%—63.03%和6.58%—49.41%, 培育期间幼虫的平均存活率31.66%, 生长率13.08 μm/d, 三倍体率降低了17.64%。综合来看, 高盐诱导是诱导三倍体熊本牡蛎的最优方法, 其诱导条件的最佳组合为盐度55, 起始诱导时间受精后15min, 持续诱导时间20min。研究为熊本牡蛎的三倍体诱导提供了技术支持, 对熊本牡蛎的多倍体育种具有重要的理论指导意义。     

鲇（♀）× 南方鲇（♂）杂交F1胚胎发育观察

通过人工授精获得鲇(Silurus asotus)♀×南方鲇(Silurus meridionalis)♂杂交受精卵,对杂交F1胚胎发育进行了观察和记录,以期了解杂交鲇早期发育特征并进一步验证鲇与南方鲇杂交的可行性。用0.3%胰蛋白酶液对受精卵进行脱黏处理,在Leica MZ16 A体式显微镜下进行观察和拍照。早期每隔15 min观察一次,发育至原肠胚阶段后每隔30 min观察一次。受精卵及胚体大小长度等用Leica DC500照相系统自带软件进行测量。结果表明,鲇(♀)×南方鲇(♂)杂交受精卵呈圆球形,草绿色,吸水后膨胀,卵径(2.50±0.14)mm,卵膜外形成一层较厚胶膜且黏性较强。在水温(24.5±0.6)℃的情况下,受精卵在受精后48 min形成胚盘,1 h 35 min开始卵裂,4 h 23 min进入囊胚期,7 h 22 min时达到原肠胚期,11 h 23 min发育至神经胚期,随后进入器官分化和逐渐完善的阶段,28 h 49 min开始出膜,40 h 33 min全部出膜。杂交胚胎的平均受精率为89.7%,孵化率为55.3%,初孵仔鱼畸形率为5.9%,表明南方鲇与鲇的精卵不亲和性程度较小,二者杂交可行。总体上看,鲇(♀)×南方鲇(♂)杂交F1胚胎发育时序、器官分化进程与其母本鲇基本相同,呈现"偏母遗传"特征。     

不同倍性虹鳟幼鱼对急性温度胁迫的抗氧化响应

虹鳟属于冷水性鱼类,其生存的最适温度为12~18 ℃,温度胁迫是虹鳟在夏秋季养殖过程中经常遇到的问题.枫叶鲑和硬头鳟均为品质优良的虹鳟选育种.为探讨急性温度胁迫对两种倍性虹鳟抗氧化响应的影响,本研究选取二倍体枫叶鲑和三倍体硬头鳟幼鱼,分别在13、17、21和25 ℃下进行热应激试验.在达到目标温度后的0、1、6和12 h取样,之后将受试鱼恢复至13 ℃的适宜温度下培养,并在恢复培养的1、12、24和48 h后取样,测定受试鱼肝脏中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)的活性,以及脂质过氧化物(LPO)、丙二醛(MDA)和热激蛋白70(HSP70)含量.结果表明: 枫叶鲑和硬头鳟的3种抗氧化酶活性在17 ℃组没有出现显著升高;21 ℃组枫叶鲑和硬头鳟的SOD活性在热应激期间出现显著升高,但枫叶鲑的SOD活性在恢复过程中恢复到正常水平;25 ℃组枫叶鲑和硬头鳟的SOD、CAT和GPx的活性均显著提高;但在恢复试验进行24 h后,CAT和GPx的活性恢复到正常水平.枫叶鲑在17、21和25 ℃组HSP70的产生量显著高于13 ℃组,而硬头鳟仅在21和25 ℃组HSP70的产生量显著高于13 ℃组.通过整合生物标志物响应指标值对多种抗氧化参数进行分析,发现枫叶鲑在17和21 ℃组急性温度胁迫下的抗氧化响应显著高于硬头鳟,但在25 ℃组硬头鳟的抗氧化响应高于枫叶鲑.这表明不同倍性虹鳟幼鱼在不同温度急性胁迫下的抗氧化响应有所差异.     

人工诱导兴国红鲤三倍体最佳诱导条件的研究

采用冷休克处理兴国红鲤受精卵诱导产生三倍体鱼 ,初步探讨了获得三倍体兴国红鲤的最佳诱导条件 ,即最恰当的诱导温度 ,最恰当的诱导起始时间 ,最恰当的冷休克处理时间。结果表明 ,在人工授精后 5～ 8min ,将受精卵置于 0～ 2℃的冰水中冷休克处理 1 0～ 1 5min ,再迅速回温至正常水温 ( 2 0～ 2 5℃ )的水中发育 ,可以获得 ( 70± 5) %的三倍体兴国红鲤胚胎 ,孵化率为 50 %～ 70 % ,成活率可达 4 0 %～50 %以上 ,此为采用冷休克技术人工诱导兴国红鲤三倍体的最佳条件。     

菊黄东方鲀、暗纹东方鲀及其杂交F1代肌肉营养成分的比较分析

为了了解菊黄东方鲀(Takifugu flavidus)、暗纹东方鲀(T.obscurus)及其杂交F1代的肌肉营养特征,利用生物化学方法,从每类实验样本中取9尾对其肌肉中的粗蛋白、粗脂肪、水分、粗灰分和氨基酸成分进行了测定和分析。结果显示:(1)杂交F1代在生长方面具有明显的杂交优势,与亲本之间存在着显著差异(P0.05),杂交F1代的体重为其亲本的1.48~1.77倍;(2)杂交F1代肌肉水分含量与其母本含量相近,但粗脂肪含量均较亲本少(P0.05),粗蛋白含量则与亲本差异不显著(P0.05);(3)除色氨酸和胱氨酸外,16种氨基酸均在肌肉样本中被检测到,除甲硫氨酸外,其余15种氨基酸间含量均存在着显著性差异(P0.05)。菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的总氨基酸含量最高,而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代总氨基酸含量则介于两亲本之间。对其必需氨基酸总量进行分析发现,菊黄东方鲀与其正反杂交F1代之间均存在显著性差异(P0.05),而暗纹东方鲀与其正反杂交F1代之间差异不显著(P0.05);(4)肌肉营养品质评价结果表明,菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的鲜味氨基酸含量为26.68%,明显高于双亲样本(菊黄东方鲀22.28%、暗纹东方鲀25.20%),而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代的鲜味氨基酸总量(23.30%)较其父本偏高,但低于其母本。研究结果表明,杂交东方鲀的肌肉营养综合了双亲的优良特性,特别是是菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代,拥有最高的鲜味氨基酸含量,具有推广价值。     

鲤科鱼类6个正反交人工异源三倍体胚胎的染色体变化及其发育命运

采用静水压休克保留第二极体的方法,在鲤(♀)×鲢(?)、鲫(♀)×鲢(?)、白鲫(♀)×鲢(?)和鲢(♀)×鲤(?)、鲢(♀)×鲫(?)、鲢(♀)×白鲫(?)6个正反交组合中都诱导出了异源三倍体,但只有正交鲤(♀)×鲢(?)、鲫(♀)×鲢(?)和白鲫(♀)×鲢(?)3个处理组中整倍性的异源三倍体胚胎才有可能正常发育,孵化出苗;而反交鲢(♀)×鲤(?)、鲢(♀)×鲫(?)和鲢(♀)×白鲫(?)3个处理组中的异源三倍体胚胎在发育过程中不断发生染色体排除和丢失,形成非整倍体而死亡,只有少数雌核发育二倍体鲢才能孵化出苗。结果表明,鱼类人工异源三倍体胚胎的发育命运与杂交物种间的基因组大小有关。     

木薯亲本正反交亲和力与授粉柱头的蛋白质组学分析

为研究木薯不同亲本杂交亲和力的差异,分别以华南5号、华南6号、华南7号为亲本进行正反交,统计不同组合间稔实率的差异。同时对受精柱头蛋白质进行分析,研究柱头对杂交亲和力影响的蛋白质调控水平。结果表明:SC5(♀)×SC7(♂)平均稔实率最高为26.22%,SC7(♀)×SC5(♂)平均稔实率最低为11.13%。通过双向电泳和质谱法分析母本SC5和SC7授粉柱头的蛋白质变化,得到27个差异蛋白质点,其中19个蛋白质出现上调表达,这些差异表达蛋白质分别参与碳代谢及能量代谢、分子伴侣和氨基酸代谢;而8个蛋白质下调表达,它们分别参与蛋白质合成及氢氰酸代谢;表明这些代谢途径对木薯亲和力高低有一定的影响。     

溪红点鲑的特殊动力作用

在水温(14.5±0.5)℃条件下,测定了平均体重(26.5±2.0)g(n=260)溪红点鲑(Salvelinus fontinalus)饱食和空腹状态下的耗氧率及排氨率.实验组共测定了260尾个体,空腹状态下标准耗氧率和标准排氨率分别为(175.00±8.49)mg/kg·h及(2.91±0.40)mg/kg·h;饱食后耗氧率和排氨率变化趋势均呈现迅速上升,到达最大值后再缓慢下降,然后恢复到初始水平,最大值分别为(375.93±9.73)mg/kg·h和(16.01±0.37)mg/kg·h,最大值出现时间分别为饱食后(6.0±0.3)h和(7.0±0.5)h,饱食后耗氧率和排氨率发生改变的持续时间分别为(23.0±1.5)h及(23.0±2.0)h,耗氧总增量和排氨总增量分别为(2641±137)mg及(164±10)mg.结果显示,溪红点鲑饱食排氨率的变化过程与其饱食耗氧率的特殊动力作用(SDA)具有类似的特征,表明二者在能量代谢机制上相互关联,溪红点鲑的SDA总耗能的18.8%±1.2%由蛋白质分解代谢所提供.     

中国对虾三倍体的诱发研究 1.温度休克

温度休克中国对虾(Penaeus orientalis)的受精卵,结果表明,用0、6和9℃的低温休克,温度越低或休克时间越长,对细胞分裂的抑制作用也越强,孵化率也越低,用30和33℃的高温休克,温度越高或休克时间越长,对细胞的伤害也越大,孵化率明显降低。低温和高温休克都能诱发三倍体,三倍体的最高诱发率,低温(9℃)为43.8％,高温(30℃)为32％,并获得三倍体蚤状幼体,对虾三倍体的诱发成功,为对虾的多倍体育种提供了可能。     

黄姑鱼三倍体的诱导、鉴定及其性腺发育特征

研究采用冷休克方法诱导黄姑鱼(Nibea albiflora)三倍体并进行鉴定,进一步采用组织学和生殖相关基因表达分析比较了三倍体黄姑鱼的性腺发育特征。研究结果表明:(1)在受精后2.5min以3℃进行10min的冷休克处理,冷休克处理组的受精率和孵化率分别为(70.31±4.49)%和(21.5±6.63)%,其受精率和孵化率显著低于二倍体对照组;(2)经流式细胞仪倍性检测和染色体核型分析发现,三倍体的DNA含量为二倍体的1.5倍,染色体数目为72条,而二倍体的染色体数目为48条,三倍体的比例为100%;(3)三倍体的生殖腺指数显著低于二倍体,进一步通过组织切片观察发现三倍体的精巢和卵巢发育较二倍体滞后,在12月龄时,二倍体精巢和卵巢处于Ⅴ期,而三倍体精巢和卵巢分别处于Ⅲ期和Ⅰ期;(4)三倍体精巢的dmrt1和vasa基因及三倍体卵巢的cyp19a基因表达量均显著低于二倍体(P<0.05);三倍体卵巢的vasa基因表达量也比二倍体低(P>0.05)。综上结果表明:研究通过冷休克处理成功诱导了黄姑鱼三倍体;三倍体的性腺发育较二倍体滞后,育性明显降低。     

Proteome Profiles of Head Kidney and Spleen of Rainbow Trout (Oncorhynchus Mykiss)

The head kidney and spleen are major lymphoid organs of the teleost fish. The authors identify proteome profiles of head kidney and spleen of rainbow trout (Oncorhynchus mykiss) using a shotgun proteomic approach. Gene ontology annotation of proteins is predicted using bioinformatic tools. This study represents detailed proteome profiles of head kidney and spleen of rainbow trout, with a total of 3241 and 2542 proteins identified, respectively. It is found that lymphoid organs are equipped with a variety of functional proteins related to defense, receptor, signal transduction, antioxidant, cytoskeleton, transport, binding, and metabolic processes. The identified proteome profiles will serve as a template for understanding lymphoid organ functions in salmonids and will increase the amount of spectra information of rainbow trout proteins in the public data repository PRIDE. This data can be accessed via ProteomeXchange with identifiers PXD008473 and PXD008478.     

Diet overlap among non‐native trout species and native cutthroat Trout (Oncorhynchus clarkii) in two U.S. ecoregions

The invasion of freshwater ecosystems by non‐native species can constitute a significant threat to native species and ecosystem health. Non‐native trouts have long been stocked in areas where native trouts occur and have negatively impacted native trouts through predation, competition, and hybridization. This study encompassed two seasons of sampling efforts across two ecoregions of the western United States: The Great Basin in summer 2016 and the Yellowstone River Basin in summer 2017. We found significant dietary overlaps among native and non‐native trouts within the Great Basin and Yellowstone River Basin ecoregions. Three orders of invertebrates (Ephemeroptera, Trichoptera, and Diptera) composed the majority of stomach contents and were responsible for driving the observed patterns. Great Basin trout had higher body conditions (k), and non‐native Great Basin trout had higher gut fullness values than Yellowstone River Basin trout, indicating a possible limitation of food in the Yellowstone River Basin. Native fishes were the least abundant and had the lowest body condition in each ecoregion. These findings may indicate a negative impact on native trouts by non‐native trouts. We recommend additional monitoring of native and non‐native trout diets, regular invertebrate surveys to identify the availability of diet items, and reconsidering stocking efforts that can result in overlap of non‐native fishes with native cutthroat trout.     

Metabolism and mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine in Chinese hamster ovary cells

The mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine (THFMP) have been studied in Chinese hamster ovary (CHO) cells in tissue culture. THFMP is relatively unstable in physiological buffers, being facilely converted to 6-mercaptopurine (6-MP) even in the absence of cells. Consequently, THFMP undergoes metabolic conversions characteristic of 6-MP, namely formation of 6-thioIMP and incorporation into DNA as 6-thioguanine (6-TG) nucleotide. A number of purines are capable of preventing the toxicity of THFMP in wild-type cells in a manner similar to that of 6-MP. However, exogenous purines and pyrimidines did not prevent the toxicity of THFMP to cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase). Cells lacking HGPRTase were 20–40-fold resistant to 6-TG and 6-MP but were only 2–4-fold resistant to THFMP. Furthermore, the time-course for killing CHO cells deficient in HGPRTase was different from that in wild-type cells containing the enzyme. There was no apparent effect of THFMP on the utilization of precursors for DNA, RNA or protein synthesis in the enzyme-deficient mutant cell line. The results suggest that THFMP is converted non-enzymatically to 6-MP and shares its mechanisms of action in wild-type cells containing HGPRTase, i.e., inhibition of de novo purine biosynthesis and incorporation into DNA as 6-TG nucleotide. However, the mechanism of action of THFMP in cells lacking HGPRTase is probably unique and is presently unknown.     

Rhamnose-binding Lectins from Steelhead Trout (Oncorhynchus mykiss) Eggs Recognize Bacterial Lipopolysaccharides and Lipoteichoic Acid

The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.     

Cryopreservation of Rainbow Trout (Oncorhynchus mykiss) Blastomeres: Influence of Embryo Stage on Postthaw Survival Rate

The cryopreservation of isolated fish blastomeres is likely to provide a valid alternative to embryo cryopreservation, the results of which are still discouraging. A repeatable technique for the cryopreservation of rainbow trout blastomeres has been established and the effect of embryonic developmental stage on freezing tolerance evaluated. Embryos at Ballard 6A, 6B, and 6C stages were dechorionated and left to dissociate in a Ca²⁺- and Mg²⁺-free medium. Cryoprotection was provided by step-wise addition of 1.4 M 1,2-propanediol. Cells were loaded into the middle of 250-μl straws and slowly frozen to −80°C before being plunged into LN₂. A low thawing rate was adopted, followed by step-wise removal of the cryoprotectant. Morphological evaluation was by microscopy and video recording. Metabolic activity and survival rate were determined by FDA and PI staining, by recovery of the ability to reassociate after 24 h culture in Leibovitz L15 + 2% Ultroser, and by measuring DNA synthesis in 6B cells by the method of BrdU incorporation. Survival rates were 53 ± 9.3, 88 ± 1.7, and 95 ± 0.5% for stage 6A, 6B, and 6C cells, respectively. While 6A cells reassociated into clumps of cells, 6B and 6C cells formed holoblastic morulas in 24 h; proliferation of 6B cells was comparable to fresh control cells. The relationship between freezing tolerance and the physiological events occurring during early embryonic development is discussed in light of these results and conclusions are drawn that envisage the transfer of frozen-thawed blastomeres into recipient embryos.     

Cryopreservation of Rainbow Trout (Oncorhynchus mykiss) Blastomeres: Influence of Embryo Stage on Postthaw Survival Rate

The cryopreservation of isolated fish blastomeres is likely to provide a valid alternative to embryo cryopreservation, the results of which are still discouraging. A repeatable technique for the cryopreservation of rainbow trout blastomeres has been established and the effect of embryonic developmental stage on freezing tolerance evaluated. Embryos at Ballard 6A, 6B, and 6C stages were dechorionated and left to dissociate in a Ca2+- and Mg2+-free medium. Cryoprotection was provided by step-wise addition of 1.4 M 1,2-propanediol. Cells were loaded into the middle of 250-μl straws and slowly frozen to -80 degreesC before being plunged into LN2. A low thawing rate was adopted, followed by step-wise removal of the cryoprotectant. Morphological evaluation was by microscopy and video recording. Metabolic activity and survival rate were determined by FDA and PI staining, by recovery of the ability to reassociate after 24 h culture in Leibovitz L15 + 2% Ultroser, and by measuring DNA synthesis in 6B cells by the method of BrdU incorporation. Survival rates were 53 +/- 9.3, 88 +/- 1.7, and 95 +/- 0.5% for stage 6A, 6B, and 6C cells, respectively. While 6A cells reassociated into clumps of cells, 6B and 6C cells formed holoblastic morulas in 24 h; proliferation of 6B cells was comparable to fresh control cells. The relationship between freezing tolerance and the physiological events occurring during early embryonic development is discussed in light of these results and conclusions are drawn that envisage the transfer of frozen-thawed blastomeres into recipient embryos. Copyright 1998 Academic Press.     

Trout hepatocyte culture: Isolation and primary culture

Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×10⁶ liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep ₄₅₀ activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep ₄₅₀ activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture. This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support Grant 5507RR05700010.     

Fatty‐Acid Profile of Total and Polar Lipids in Cultured Rainbow Trout (Oncorhynchus mykiss) Raised in Freshwater and Seawater (Croatia) Determined by Transmethylation Method

Fatty acids from total lipids and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in seawater (SW) and freshwater (FW) were identified and quantified from the muscle samples in January, April, and July. The highest total lipid and polar lipid amounts were found in April. July contents of total lipids were low, but percent of the polyunsaturated fatty acids (PUFAs) was high in SW and FW environment (particularly n‐3 PUFAs). Variety of 17 fatty acids was identified by GC‐FID after transmethylation. The predominant fatty acids in rainbow trout from SW and FW were: docosahexaenoic acid among n‐3 PUFAs, palmitic acid among saturated fatty acids (SFAs), and oleic acid among monounsaturated fatty acids (MUFAs). Appreciably higher n‐3/n‐6 ratio was found in total lipids in April (6.40, FW fish) and in polar lipids in July (18.76; SW fish). High n‐3/n‐6 ratio in total lipids and polar lipids of rainbow trout from SW and FW, besides beneficial n‐3/n‐6 ratio in the commercial fish food, could be characteristic for the local environmental conditions (Croatia).     

The In Vitro and In Vivo Inhibitory Effects of Some Sulfonamide Derivatives on Rainbow Trout (Oncorhynchus Mykiss) Erythrocyte Carbonic Anhydrase Activity

The in vitro and in vivo inhibitory effects of 5-(3α, 12α-dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α, 7α, 12α-trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α, 7α, 12α-triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3) and acetazolamide on rainbow trout (Oncorhynchus mykiss) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5?EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO₂-Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I₅₀ value for the sulfonamides (1), (2) and (3) and acetazolamide were 0.83, 0.049, 0.82 and 0.052?μM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides (1), (2) and (3) and acetazolamide.