A practical guide for assigning sex and stage of maturity in sturgeons and paddlefish

The goal of this paper is to improve the assignment of sex and stage of maturity for sturgeons and paddlefish by providing both an overview of the gonadal stages of maturity and general guidelines and training needs for the four most commonly used techniques to assign sex and stage of maturity including ultrasound, endoscopy, plasma sex steroid analysis, and biopsy of the gonads via celiotomy. Sturgeons and paddlefish do not express external sexual dimorphism, which can make assignment of sex and stage of maturity challenging. Correct assignment of sex and stage of maturity is important for management of wild populations as well as for aquaculture, whether for conservation or commercial production. Selecting a technique to use when assigning sex and stage of maturity will depend on a number of factors; a comparison among these techniques is provided in this review, including tradeoffs, to help assist in technique selection based on specific research or production goals. The use of more than one technique may be beneficial to determine error rates associated with a single technique. This review is intended to serve as a practical resource when assigning sex and stage of maturity in field or laboratory settings in addition to stressing the importance of correctly identifying sex during research and in the management of populations.     

Dynamic Dialysis: An Efficient Technique for Large-Volume Sample Desalting

Dialysis is a well-known technique for laboratory separation. However, its efficiency is commonly restricted by the dialyzer volume and its passive diffusion manner. In addition, the sample is likely to be precipitated and inactive during a long dialysis process. To overcome these drawbacks, a dynamic dialysis method was described and evaluated. The dynamic dialysis was performed by two peristaltic pumps working in reverse directions, in order to drive countercurrent parallel flow of sample and buffer, respectively. The efficiency and capacity of this dynamic dialysis method was evaluated by recording and statistically comparing the variation of conductance from retentate under different conditions. The dynamic method was proven to be effective in dialyzing a large-volume sample, and its efficiency changes proportionally to the flow rate of sample. To sum up, circulating the sample and the buffer creates the highest possible concentration gradient to significantly improve dialysis capacity and shorten dialysis time.     

New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent.

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.     

A source of error in equilibrium dialysis

Equilibrium dialysis is often used to study the binding of steroids to proteins. With this technique it is customary to determine the percent bound and unbound steroid in the sample, the affinity constant for the steroid-protein binding reaction, and the concentration of binding sites on the protein. Investigators have used many different ratios of dialysis buffer to sample volumes in their experiments assuming that the equilibrium in the post-dialysis sample was the same as existed before dialysis. Chemical equilibrium expressions for the system before and after dialysis indicate that during dialysis the concentration of steroid in the sample decreases resulting in a new equilibrium in which the percent bound and unbound are different from the original sample. The magnitude of the difference between the pre- and post-dialysis systems is proportional to the ratio of dialysis buffer to sample volumes. Accurate values for the affinity constant and binding site can be obtained only if this change in the equilibrium is considered.Experimental verification of the application of these principles was made in an equilibrium dialysis study of testosterone-albumin binding.     

Flurbiprofen release from eudragit RS and RL aqueous nanosuspensions: a kinetic study by DSC and dialysis experiments

The present work investigated the release of Flurbiprofen (FLU) from Eudragit RS100 (RS) and Eudragit RL100 (RL) nanosuspensions to a biological model membrane consisting of Dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV). This release was compared with those observed from solid drug particles as well as with dialysis experiments. Nanosuspensions were prepared by a modification of Quasi-Emulsion Solvent Diffusion technique. Drug release was monitored by the Differential Scanning Calorimetry (DSC). FLU dispersed in MLV affects the transition temperature (T(m)) of DMPC liposomes, causing a shift towards lower values. The temperature shift is modulated by the drug fraction present in the aqueous lipid bilayer suspension. DSC was also performed, after increasing incubation periods at 37 degrees C, on suspensions of blank liposomes added to fixed amounts of unloaded and FLU-loaded nanosuspensions, as well as to powdered free drug. T(m) shifts, caused by the drug released from the polymeric system or by free-drug dissolution during incubation cycles, were compared with those caused by free drug increasing molar fractions dispersed directly in the membrane during their preparation. These results were compared with the drug release and were followed by a classical dialysis technique. Comparing the suitability of the 2 different techniques in order to follow the drug release as well as the differences between the 2 RL and RS polymer systems, it is possible to confirm the efficacy of DSC in studying the release from polymeric nanoparticulate systems compared with the "classical" release test by dialysis. The different rate of kinetic release could be due to void liposomes, which represent a better uptaking system than aqueous solution in dialysis experiments.     

透析培养

本讨论了有效利用透析技术从发酵液中及时转移低分子杂质混合物,从而获得高密度发酵细胞的方法,章从反应系统、工艺策略、膜相关性能、应用我、生产性放大等方面说明了利用透析技术以达到高浓度细胞发酵的有效性和可靠性。透析技术不仅克服了微孔过滤和超滤中存在的膜孔堵塞弊端,而且如果应用“营养分离”补策略,还可以防止营养物质的损失而使培养基被高效利用,在实验条件下,透析培养的潜力通过两种反应模型进行演示：内置     

3R和替代方法是否进行了有效的探究（英文）

Although the publication of peer-reviewed scientific papers is the major way in which scientists communicate with one another,these papers often lack essential information about the conditions under which the animals lived and how they were treated during the experiments.Without this information it may be impossible to evaluate the studies,replicate them in another laboratory,or use them to advance the 3Rs in other animal experiments.This paper gives some advice on how the reporting of animal research can be improved.     

A novel dialysis procedure for the crystallization of proteins

Various dialysis methods are commonly employed for the crystallization of proteins. Typical procedures include the use of dialysis bags, dialysis buttons or Zeppezauer microdiffusion cells. The general principle involved is that the protein solution is gradually brought to a point of supersaturation by imposing a gradient of ionic strength or organic solvent concentration across the wall of the dialysis membrane. However, in some cases, the imposition of this gradient across the dialysis membrane can result in the formation of a large number of crystal nucleation sites, thereby giving rise to a reduction in the maximum size of the crystals which can be obtained. A novel 'double-dialysis' procedure which incorporates a second dialysis membrane, thus reducing the rate of equilibration in the crystallization experiment, has been developed in our laboratory. The system has been employed successfully on the delta toxin of Staphylococcus aureus resulting in a useful increase in crystal size. A more quantitative analysis of the technique has been carried out on rat liver malic enzyme. The results of a limited series of crystallization trials with this protein have shown that employment of the 'double-dialysis' technique allows a fine control of the rate of crystal nucleation and therefore provides a mechanism for the controlled growth of large crystals.     

New cloning vectors and techniques for easy and rapid restriction mapping

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Uptake of 86Rb by human erythrocytes: modification of the method and applications]

In this study we applied a method generally used for the study of Na+,K(+)-ATPase, as well as other systems of potassium transport, which makes use of a rubidium isotope (86Rb) as analogue of the potassium and is known as uptake of the 86Rb. This method proved to be particularly sensitive and versatile for kinetic studies of this pump system, allowing to assess possible alterations. Its application in the study of sodium and potassium transport in erythrocytes of uremic subjects in extracorporeal dialysis made it possible to reveal certain alterations due both to pump-dependent and pump-independent uptake. In fact, the results show the hypothesis of restoration of Na+,K(+)-pump activity for elimination during dialysis of one or more inhibitor present in the uremic plasma. Furthermore, a reduction in aspecific flows was noted which could be the result of more generalized damage of the membrane.     

Gene conversion of immunoglobulin variable regions in mutagenesis cassettes by replacement PCR mutagenesis.

A technique, Replacement PCR Mutagenesis, was developed to replace one immunoglobulin variable region (V) in a M13 phage cassette with a different, homologous V. This allows the use of the same mutagenesis and subsequent expression vectors for many V regions or V segments. The method combines PCR of V fragments and in vitro mutagenesis. Primers homologous to 3' and 5' ends of both V regions initiate PCR synthesis of the V DNA fragment (donor) that will replace the V region (recipient) in M13. Donor V PCR DNA may originate from mRNA, cloned V genes or genomic templates. The donor V PCR DNA is denatured and annealed to the M13 cassette containing the recipient V to be supplanted. The second strand is synthesized, transfected into bacteria and mutant plaques selected by hybridization. Since restriction sites in primers are not required, altered primer-encoded amino acids are avoided. Further, the PCR donor piece can be of any length if it shares homology with the recipient gene. This allows construction and expression of complete gene replacements and chimeras. This method is also applicable to V "humanization" and studying sets of homologous genes containing polymorphic or evolutionary disparities. The potential uses of the technique are discussed.     

Behavioural assessment of the effects of tumour growth in rats and the influence of the analgesics carprofen and meloxicam

Very little is known concerning the occurrence of pain in cancer research models. We wished to establish whether a behaviour-based approach, originally developed to assess postoperative pain, could be used to determine positive effects of the analgesics carprofen and meloxicam in rats that might be experiencing pain during tumour development in an orthotopic model of bladder cancer. An invasive but non-metastatic rat bladder cancer cell line was surgically implanted into the bladder wall of 57 inbred Fisher344 rats. The rats underwent daily clinical assessments. When clinical signs consistent with chronic pain were apparent, behavioural data were collected from 44 animals during 2 x 10 min periods, immediately before and one hour after a subcutaneous injection of either physiological saline (0.9%; 0.2 ml/100 g), carprofen (5 mg/kg) or meloxicam (2 mg/kg). Treatment-associated behaviour changes were then compared between groups. The lack of active behaviour, both before and after each treatment, was consistent with established clinical signs of pain. The rats were so inactive following the treatment that the behavioural technique we had previously developed was of comparatively little use in determining either pain severity or analgesic efficacy. One very prominent effect, however, was an increase in ventral abdominal licking in the control (saline) group. As this was absent in rats given meloxicam or carprofen, and has previously been considered to indicate pain emanating from damaged tissue, it was concluded that the analgesic-treated rats gained at least some benefit from the drug treatments, but it was not possible to gauge the extent of this. Handling for examination or treatment may have intensified pain in rats in the control group, and so this should be avoided whenever possible. It is likely that post-surgical pain differs markedly from cancer pain, so a different set of behavioural markers may be needed to assess it effectively. More intensive behaviour monitoring may help to develop a suitable technique for detecting the onset of, and assess the severity of pain that may occur during tumour development.     

Haemofiltration for profound dialysis-induced hypotension: removal of sodium and water without blood-pressure change.

Two patients with profound dialysis-induced hypotension were seen, in both of whom sequential ultrafiltration and haemodialysis failed to alleviate their symptoms; in one bicarbonate dialysis similarly produced no improvement. The hypotension was frequently severe enough to necessitate premature termination of the dialysis. Haemofiltration was associated with almost total resolution of symptoms, adequate biochemical control of uraemia, and satisfactory removal of weight gain between dialysis sessions. Profound hypotension during dialysis may become more common as older patients enter dialysis programmes; haemofiltration is a valuable technique in such cases.     

Peritoneal Dialysis in Chronic Renal Failure

The long-term results of intermittent peritoneal dialysis in long-term treatment of renal disease have yet to equal those of intermittent hemodialysis. However, further exploration and refinement of this technique is justified.Performed in acute stages of disease, both peritoneal dialysis and hemodialysis relieve the symptoms of uremia and specifically “buy time” for the patient so that proper medical or surgical therapy may be instituted. In acute situations, peritoneal dialysis is the procedure of choice, and is an important adjunct to more conventional treatment for chronic renal disease. It may be useful sometimes even in chronically hemodialyzed patients—for example, when the hemodialysis cannula for one reason or another is inaccessible because of clots, replacement, or infection. It is especially valuable when the hemorrhagic complications of uremia contraindicate hemodialysis treatment.Its use in chronic uremia remains sharply limited in time, but for brief periods chronic peritoneal dialysis appears to be a reasonably satisfactory means of prolonging life while awaiting homotransplant or decision for maintenance hemodialysis therapy.     

The hibernation cycle and related changes in the brown fat tissue of Citellus lateralis

The hibernating habits of Citellus lateralis under standard laboratory conditions are described. It has been shown that it is possible to use a biopsy technique to investigate the brown fat tissues without serious disturbance to the hibernation pattern. This technique was used to sample brown and white fat tissues in the same animal at various seasons of the year. Histological studies did not reveal any changes in morphology or lipid composition which could be related purely to the hibernating season, however, during the short periods of arousal a large proportion of the brown fat cells appeared to be partially depleted of neutral fat. At this time a large increase in Luxol fast blue staining was always observed, which may be due to unmasking of tissue phosphatides.     

红莲型水稻与细胞质雄性不育相关mtDNA片段的分离及序列测定

应用AFLP分析技术,比较了红莲型水稻雄性不育系和保持系线粒体DNA,找到一不育系特异条带TA1。以该条带DNA为探针与不育系、保持系以及F1杂种线粒体总RNA杂交,结果显示该片段黄化苗期三者均转录一个RNA分子,但转录产物分子量各不相同,说明三者该片段阅读框架不同。经测序,该片段长202bp,序列内含密码子ATG、ATT、AGA、AGG以及正向重复序列5’TGTAC3’、5’ATTATTTT3’和倒重复序列5’GGGAAACA3’。上述特点表明该片段可能是某一蛋白编码序列,并可能与红莲型水稻雄性不育性状形成有关。     

The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors

From previous work it was known that U3 RNA is hydrogen bonded to nucleolar 28 S to 35 S RNA and can be covalently crosslinked to RNA of greater than 28 S by irradiation in vivo with long-wave ultraviolet light in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT psoralen). Here we use a novel sandwich blot technique to identify these large nucleolar RNA species as rRNA precursors and to map the site(s) of crosslinking in vivo. The crosslink occurs between one or more residues near the 5' end of U3 RNA and a 380 nucleotide region of the rat rRNA external transcribed spacer (ETS1). We have sequenced this region of the rat ETS and we show that it includes an RNA-processing site analogous to those previously mapped to approximately 3.5 kb upstream from the 5' end of mouse and human 18 S rRNAs.     

Mannitol

Mannitol may be useful clinically both as a diuretic and as an obligate extracellular solute. As a diuretic it can be used to treat patients with intractable edema states, to increase urine flow and flush out debris from the renal tubules in patients with acute tubular necrosis, and to increase toxin excretion in patients with barbiturate, salicylate or bromide intoxication. As an obligate extracellular solute it may be useful to ameliorate symptoms of the dialysis disequilibrium syndrome, to decrease cerebral edema following trauma or cerebrovascular accident, and to prevent cell swelling related to renal ischemia following cross-clamping of the aorta. Largely unexplored uses for mannitol include its use as an osmotic agent in place of dextrose in peritoneal dialysis solutions, its use to maintain urine output in patients newly begun on hemodialysis, and its use to limit infarct size following acute myocardial infarction.