On the presence of proteolytic activity in glycosaminoglycan-degrading enzyme preparations

A solid-phase protease assay has been used to screen different commercial preparations of glycosaminoglycan-degrading enzymes for the presence of proteolytic activity. Proteases cannot be detected in preparations of testicular hyaluronidase and of chondroitinase at the concentration used for histochemical purposes. Commercial Streptomyces hyaluronidase contains proteolytic contaminants detectable at the concentration used for histochemistry. At higher concentrations, all preparations appear to be contaminated with proteases. The results obtained using this assay suggest that addition of a mixture of proteinase inhibitors containing N-ethylmaleimide, EDTA, pepstatin, and phenylmethanesulfonylfluoride or soybean trypsin inhibitor has little effect on the proteolytic activity of the glycosaminoglycan-degrading enzyme preparations, irrespective of the pH used. Moreover, the use of EDTA in this mixture is questionable. This study also describes two testicular hyaluronidase preparations that may be particularly useful in functional studies of the living organism, as they are only slightly contaminated.     

Human interferon for clinical trials: removal of pyrogen by a simple two-step procedure

Commonly used preparations of human interferon are pyrogenic when used in humans for clinical trials. We describe a simple two-step procedure for the purification of human fibroblast interferon, employing Blue Sepharose chromatography and high-speed centrifugation. Such preparations are devoid of pyrogenic activity in a rabbit test system and are much more suitable for use in human clinical trials than previously used preparations.     

NEWER HORMONAL PREPARATIONS

A review of the present status of various hormonal substances is presented. The pituitary preparations include various growth hormones (human and beef), still used only experimentally, thyrotropic hormone, used mainly for testing thyroid function, corticotropin—widely used—and gonadotropic hormone.Thyroid, thyroxin and triiodothyronine preparations are considered, with USP thyroid still being most useful. Glucagon may be of some use in terminating hypoglycemia — tolbutamide is now used in many older diabetic persons. New adrenal cortical steroids are still appearing and show variation in effects; cortisone or hydrocortisone remain relatively inexpensive. Many combinations are available.The newest addition to available male hormone preparations is fluoxymesterone which is anabolic in smaller dosage than the older forms.Several new long acting preparations of androgens, estrogens and progesterone are available, and many ingenious combinations are presented.     

Supplementary quality controls of plasma fractions for manufactures and consumers

As a consequence of the increasing use of plasma fractions, there is an ever-growing abundance of such preparations. These preparations may be divided into three main groups: clotting factors, immunoglobulins, and plasma protein solutions. Frequency of component-induced side-effects are inversely proportional to those components' quality. Pharmacopoeas presently require only the following quality criteria for most plasma preparations: declaration of sort and concentration of the active component and stabilizers, sterility, and the absence of pyrogens. Of great importance is the origin of the raw material and the methods used to harvest the plasma, as well as the molecular condition of the proteins. While we can assume that presently-available preparations meet minimum requirements, it can be demonstrated that similar preparations can in no case be considered equivalent when immunoelectrophoresis, ultracentrifugation and gel filtration are used as additional quality control tests. Especially immunoelectrophoresis and gel filtration are tests easily available to the clinician to help him detect and thus select the optical product.     

Crush preparations of lesions of the central nervous system. A useful adjunct to the frozen section

A series of 32 cases in which crush preparations were used in addition to frozen sections for the rapid diagnosis of lesions of the central nervous system (CNS) is presented. The cytopathologic features in crush preparations of astrocytomas, glioblastomas multiforme and a pituitary adenoma are described. Excellent preservation of cellular detail was seen in the crush preparations. Frozen sections lacked cytologic detail but provided a better view of the tissue architecture. The crush preparations yielded the correct diagnosis in 29 of the 32 cases. In the other three, a secondary component of the neoplasms (oligodendroglioma and fibrosarcoma) was identified only in the paraffin sections. Use of both frozen sections and crush preparations is recommended for all cases in which an immediate diagnosis of a CNS lesion is required.     

Standardization of preparations intended for the serodiagnosis of arboviruses

As the result of our research work, 3 reference preparations have been first obtained and studied in accordance with all requirements of biological standardization. These preparations are the national standard of yellow fever antiserum and immune ascitic fluids (IAF) used as reference reagents: IAF to tick-borne encephalitis virus and IAF to Japanese encephalitis virus. The new preparations are stable, possess sufficient specific activity and can be used as standard preparations for the identification of the above-mentioned viruses.     

Brief Communication Commercial Pha Preparations Contain Different Mitogenic Components

Three commercially available purified phytohaemagglutinin (PHA) preparations have been examined both chemically and biologically. Marked differences were observed in both composition and mitogenic effect on lymphocytes; the existence of at least two distinct mitogenic fractions in PHA was confirmed. A number of commercial preparations of PHA have become available for use as mitogens in lymphocyte cultures. Since there is no cross-standardization of biological activity between these preparations, workers have used their own discretion about which to use and whether further purification is desirable. Three commercial purified PHA preparations have been examined, and marked biological and chemical differences have been found.     

Cautionary note on the use of [methyl-3H]thymidine to measure rates of chloroplast DNA synthesis in Chlamydomonas reinhardtii

Commercial [methyl-3H]thymidine preparations tested here contain about a 0.2% contaminant which is rapidly incorporated into Chlamydomonas DNA. This contaminant obscures the measurement of the rate of chloroplast DNA synthesis when methyl-labeled preparations are used. Such contaminants are not present in ring-labeled (either 3H or 14C) thymidine preparations. In ring-labeled thymidine preparations, a slower incorporation rate commensurate with cell density is observed. These slower, long-term incorporation kinetics would be expected for the utilization of bona fide thymidine into chloroplast DNA.     

噬菌体分离纯化技术研究进展*

噬菌体应用领域十分广泛,因此在制备噬菌体过程中,需采用不同的技术或几种技术相结合的方法来获得具有不同纯度的噬菌体制剂。常用的技术主要包括沉淀、过滤和离心。近年来,色谱技术、场流分流技术和电泳技术等的应用,为制备噬菌体制剂提供了新的方向。     

Enzymatic characterization of the polynuclear aromatic hydrocarbons activating rat-liver preparations used in the mutagenicity test of Ames

Stability studies were performed on the mono-oxygenase system involved, in particular, in the activation of polynuclear aromatic hydrocarbons (PAHs) present in rat-liver preparations used in the Ames mutagenicity test. The results indicated a good stability of the spectral response of the cytochrome-P-450 system, but a much lower stability of its enzymatic activities measured with various substrates, thus showing the inadequacy of the spectral response to characterize the PAH mono-oxygenase activity of the preparations. Epoxide hydrolase activity was found to be stable. Various mono-oxygenase activities were measured in preparations induced with phenobarbital, 3-methylcholanthrene or Aroclor 1254. The activities of two enzymes, benzo[a]pyrene hydroxylase and ethoxyresorufin-O-dealkylase, were found suitable to characterize the capacity of the preparations to metabolize PAH to mutagens. The efficiency of the same preparations to promote the mutagenicity of benzo[a]pyrene and aflatoxin B1 in the Ames test was determined. There was an excellent general correlation between the efficiencies for mutagenic activation of the preparations and the two enzymatic activities mentioned above. Determination of ethoxyresorufin-O-dealkylase (or benzo[a]pyrene hydroxylase) and benzo[a]pyrene 4,5-oxide hydrolase activities is proposed for characterizing the rat-liver preparations used in the Ames test.     

Electron microscopic examination of common red cell ghosts: cytoplasmic contamination.

Red blood cell ghost preparations are often cited as providing unequivocal or convincing evidence for the active transport of solutes from a solution of low concentration across a membrane to a solution of higher concentration. Electron microscopic examination of the more widely used ghost preparations show that a considerable quantity of cytoplasmic macromolecules (including hemoglobin) remain within the treated red blood cells. That is, many of the ghost preparations are not hollow membrane perparations. It is concluded that the problem of active solute transport in red blood cell ghost preparations should be reexamined. Furthermore, experiments with ghost preparations purporting to demonstrate active transport should include electron photomicrographs of the preparation utilized.     

Impact of virus preparation quality on parvovirus filter performance

Virus‐removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus‐removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010 ). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations. Biotechnol. Bioeng. 2013; 110: 229–239. © 2012 Wiley Periodicals, Inc.     

Food interactions with once-a-day theophylline preparations: a review

At present, theophylline is used predominantly as sustained-release dosage forms. Since the mid-seventies many such products have been introduced and have found huge application for use with a dosage interval of 12 hr ('twice-a-day' preparations). Since 1983 theophylline has also been available as preparations that can be given with an interval of 24 hr ('once-a-day' preparations). The release of theophylline from sustained-release dosage forms can be influenced (either increased or decreased) by concomitant intake of food. Obviously, ultra-slow-releasing products are most vulnerable to food effects. With some preparations the composition of the meal, especially its fat content, determines the degree of the food effect. The effect of meal timing and content on once-a-day theophylline preparations must be known since rather large doses are ingested all at a single time. If food can alter the release of theophylline in an unexpected manner from ultra-slow preparations, drug effectiveness may be impaired if release is inhibited or toxicity might result if sudden release of drug occurs. Herein, information about food interaction with once-a-day theophylline preparations is reviewed as this topic is important both for clinicians as well as those concerned with chronopharmacologic investigations of such medications.     

THE USE OF MICROBIAL MEMBRANES TO ACHIEVE ANAEROBIOSIS

Several years ago, it was observed that sterile microbial membrane preparations stimulated recovery of certain radiation-injured bacteria. Later it was noted that these same preparations reduce dissolved oxygen to water in a variety of environments, including bacteriological media. This reduction of oxygen is an enzymatic process and is influenced by parameters such as temperature, pH, and the availability of specific oxidizable substrates. Oxygenreducing membrane preparations can be made from several different bacterial species. When added to liquid or solid bacteriological media, membrane preparations rapidly produce and maintain anaerobic conditions favorable for the growth of a wide variety of oxygen-sensitive microorganisms. When used with a specifically designed disposable dish, membrane preparations allow the development of colonies of many anaerobic microorganisms on the surface of agar without the use of anaerobic hoods or other devices. In addition to providing conditions suitable for the growth of anaerobes, membrane preparations stimulate recovery of heat and cold injured bacteria of several different genera including facultative organisms. These results are reminiscent of the early observations regarding the recovery of radiation-injured bacteria. In addition to their usefulness in microbiology, oxygen-reducing membrane preparations have the potential for protecting a wide variety of oxygen-sensitive organic compounds.     

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures

Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.     

The assessment of viability in isolated rat hepatocytes.

Isolated rat hepatocytes are used in many metabolic studies, but the viability of these cell preparations is often not adequately established. The present study shows that ATP content is a more reliable index of metabolic viability than trypan blue exclusion. At some of the low trypan blue exclusion levels quoted in the literature, a high percentage of cell preparations is likely to be nonviable by the criterion of ATP content. We suggest that ATP content measured on initial cell preparations and at the end of all incubation procedures is essential for establishing cell viability for metabolic studies on isolated hepatocytes.     

Protein synthesis in muscles from normal and dystrophic hamsters.

Homogenates of hindleg muscle were obtained from control and dystrophic male hamsters, 30 and 190 days of age, and were used to prepare the postmicrosomal pH5-supernatant fraction. The activity of this fraction in the incorporation of [14C]phenylalanyl-tRNA into peptides was increased in the dystrophic-muscle preparations. No such increase was found in brain or liver preparations from dystrophic hamsters. The increased capacity for aminoacyl-tRNA binding that was observed in preparations from dystrophic animals is discussed.     

A spin-reduction assay for lipoxidase.

Spin-label signals are reduced when the nitroxide is present in a lipoxidase reaction mixture. This spin reduction can be used as an assay for enzyme activity in turbid systems where the conventional uv assay cannot be used. Data taken with the spin-reduction assay show that phospholipid preparations containing cholesterol are more resistant to oxidation by lipoxidase than are cholesterol-free preparations.     

Comparative evaluation of different meningococcal group-specific erythrocyte diagnostic kits in passive hemagglutination test

The comparative evaluation of the sensitivity and specificity of serogroup A, C, Y meningococcal antigenic preparations obtained by different methods was carried out by means of the passive hemagglutination test. In case of group 0 (I) human red blood cells sensitized with serogroup A and C vaccinal preparations obtained by Gotschlich's method (designated as A-1 and C-1) were used. In the other case formalin-treated sheep red blood cells sensitized with group-specific polysaccharides obtained by alcohol precipitation from the cultural fluid of group A, C and Y meningococci with subsequent heating (designated as A-2, C-2, Y) were used. Titrations with commercial immune rabbit sera showed that both variants of the antigenic preparations were similar in their specificity and sensitivity. In patients with the symptoms of meningitis the diagnostic titer was 1:40 for preparations A-1, C-1 and 1:80 for preparations A-2, C-2 and Y. The results of the examination of 164 patients (220 serum specimens) demonstrated that these preparations were of equal diagnostic value.     

In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations

Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function.