Isolation and characteristics of Klebsiella pneumoniae lysozyme factor

For the first time the preparation of K. pneumoniae antilysozyme factor has been isolated and purified. This factor, having a molecular weight of about 1,000 daltons, is oligopeptide with an oligosaccharide part. It also contains such amino acids as proline, lysine, arginine and tyrosine. The antilysozyme factor is resistant to 30-minute boiling and has no optimal pH value for its action.     

Two-day radial-arm water maze learning and memory task; robust resolution of amyloid-related memory deficits in transgenic mice

The radial arm water maze (RAWM) contains six swim paths (arms) extending out of an open central area, with an escape platform located at the end of one arm (the goal arm). The goal arm location remains constant for a given mouse. On day 1, mice are trained for 15 trials (spaced over 3 h), with trials alternating between visible and hidden platform. On day 2, mice are trained for 15 trials with the hidden platform. Entry into an incorrect arm is scored as an error. The RAWM has the spatial complexity and performance measurement simplicity of the dry radial arm maze combined with the rapid learning and strong motivation observed in the Morris water maze without requiring foot shock or food deprivation as motivating factors. With two sessions each day, 16 mice can be tested over 2 days.     

Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli

We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.     

A comparison of rapidly transported proteins in the sensory fibers of the rat and mouse sciatic nerve

Fast axoplasmic transport through the sensory fibers of the sciatic nerve has been compared in rats and mice. The use of in vitro incubation permits high levels of specific activity to be attained when labeling with [³⁵S]l-methionine. The specific activity of the transported proteins was about 10-fold greater in mice than in rats. Proteins labeled with radioactive methionine were examined after separation on polyacrylamide gels. There are no differences between mice and rats when the proteins carried by rapid transport are compared. Similarly, the proteins synthesized by the Schwann cells of these two species are not distinguishable. The dorsal root ganglia of mice, however, yield a band of radioactivity that is not seen in ganglia from rats. This band migrates with an apparent molecular weight of 31,000 daltons.     

Isolation of a plasma protein elevated during hypertension in the rat

Plasma from male rats contains a protein that is elevated during essential hypertension. This protein, termed hypertension associated protein (HAP), can be detected as a peptide that has a molecular weight of 14,000 daltons on high resolution SDS-gradient polyacrylamide gels. The native protein has now been isolated by elution from DEAE-Sepharose, carboxymethyl cellulose and by gel permeation on Ultrogel AcA44. The procedure yields 102 mg of highly purified protein from 5 ml (250 mg) of plasma in 72 h. The native protein has a molecular weight of 28,000 daltons.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Differential inactivation and separation of homologous and heterologous antiviral activity of human leukocyte interferon by a proteolytic enzyme.

Human leukocyte interferon (HuLeIF) can express its antiviral activity on both human and bovine cells. The rates of inactivation of HuLeIF by α-chymotrypsin, as expressed on human and bovine cells, are not the same: the ability to induce activity on human cells is lost significantly more rapidly than the activity detected on bovine cells; usually a margin of greater than one hundred-fold exists after α-chymotrypsin treatment. HuLeIF, when subjected to analysis on 10% SDS-PAGE, can be separated into two molecular weight species, one having apparent molecular weight of approximately 21,000 daltons, the other 18,000 daltons. A more rapidly migrating form (molecular weight 16,500 daltons) can also be isolated, which is considerably more active on bovine cells than on human cells. α-chymotrypsin-treated samples analyzed by SDS-PAGE show a clear separation of the activities expressed on human and bovine cells. The residual activity detected on human cells is isolated only in the 21,000 component while the activity found on bovine cells is recovered only as the 16,500 dalton species.     

Immunological study of the cellular components of Pseudomonas aeruginosa. VI. The toxicity and protective properties of its mucus and its fractions]

Capsule-like mucus was obtained from two newly isolated mucoid strains of P. aeruginosa and then treated with ethanol. The mucus was fractionated by the method of differential centrifugation (at 15, 000 g for 1 h, at 105, 000 or 170, 000 g for 3 h) and gel chromatography in columns packed with Sepharose 4B. The sediment fractions contained 30--80% of high molecular polysaccharide protein (peptide) mucus components which were toxic for mice and protected 25--77% of rats against experimental P. aeruginosa infection. The supernatant fluid fractions contained 60--80% of predominantly protein components with molecular mass between 20,000 and 60,000 daltons. These mucus components were slightly toxic for mice and rats and protected 80--100% of rats against P. aeruginosa infection.     

Molecular forms of immunoactive atrial natriuretic peptide released from cultured rat atrial myocytes

Primary cultures of atrial myocytes were prepared from newborn rats and maintained for 8 days in complete serum-free medium. The culture content of immunoactive atrial natriuretic peptide (ANP) increased from 10 to 25 ng/culture during this time. The cells released immunoactive ANP at a rate of 2 to 3% of culture content per hour in a linear fashion for at least 6 hours. When analyzed by gel filtration the major immunoactive material released by and contained within the cells displayed a molecular weight of approximately 15,000 daltons. The medium and cellular ANP-related peptides were further shown to be indistinguishable by reversed-phase HPLC. When the 15,000 dalton material was incubated with rat serum it was converted to ANP-related material possessing a molecular weight of approximately 3,000 daltons. These results suggest that under basal conditions, atrial myocytes release a large molecular weight form of ANP that is converted in the circulation to a low molecular weight form of ANP, which has been previously identified in plasma.     

Correlation between Production of Inflammatory Substances and Generation of Effector T Cells Mediating Delayed-Type Hypersensitivity in Mice

In vitro exposure to human serum albumin (HSA) of splenic lymphocytes from mice sensitized for delayed-type hypersensitivity (DTH) against HSA resulted in the release of substances that could induce a footpad inflammatory reaction with a maximum 6 hr after injection into normal mice. The substances were fractionated mainly in a molecular weight range of 30,000 to 70,000 daltons on Sephadex G-200. The ability of sensitized lymphocytes to produce the substances was dependent on T cells, was antigen specific, and correlated well with the ability of the lymphocytes to mediate DTH reactions. Moreover, the substances were produced efficiently by the DTH effector cell population generated in the in vitro culture system and also by the effector cell-enriched fractions on discontinuous bovine serum albumin gradients. These results suggest that the substances are produced by DTH-effector cells.     

Antigenicity of a proteolytic enzyme of Fasciola hepatica

The possibility was examined of using a haemoglobinase released during in vitro incubation of adult Fasciola hepatica for immunodiagnosis of fascioliasis. By SDS gel electrophoresis the enzyme appeared as two closely migrating bands with a molecular weight of approximately 27,000 daltons. After Western blotting the bands reacted with serum from rats infected with F. hepatica and mice infected with Schistosoma mansoni. The enzyme was therefore not a good antigen for immunodiagnosis.     

Modification of yeast uricase with polyethylene glycol: disappearance of binding ability towards anti-uricase serum

Uricase from Candida utilis was modified with activated polyethylene glycol (2-O-methoxypolyethylene glycol-4,6-dichloro-s-triazine) of molecular weight of 5,000 daltons. The modification of 43% of the total amino groups in the uricase molecule gave rise to a complete loss of the binding ability towards anti-uricase serum from rabbit. This modified uricase retained 15% of the enzymic activity of non-modified uricase.     

The fate of the biomimetic cation triaqua-mu-oxohexapropionatotrichromium(III) in rats

The synthetic biomimetic triaqua-mu-oxohexapropionatotrichromium(III) nitrate when given intravenously to rats lowers fasting blood plasma triglycerides and cholesterol concentrations; thus, the cation has the potential to serve as a therapeutic agent. Its ability to function in vivo presumably is dependent on its ability to mimic the action of the natural, bioactive, chromium-binding oligopeptide chromodulin in stimulating insulin receptor kinase activity. Consequently, the cation should be incorporated into insulin-sensitive cells intact. Thus, the fate of the 51Cr-labeled complex during the first 24 h after injection in tissues, blood, urine, and feces was followed. The complex appears to be readily incorporated into tissues and cells. In hepatocytes, the cation is efficiently transported into microsomes where its concentration reaches a maximum in approximately 2 h.     

Hypometabolic brain peptide from vertebrates capable of torpor

One or more agents found in extracts of brains from estivating lungfish and hibernating ground squirrels depress oxygen consumption and colonic temperature when injected intravenously into mice. These observations from different laboratories confirm initial similar reports using rats as subjects. Comparison with rat brain extract injections indicate that the hypothermia was not solely a nonspecific response to foreign substance in the blood. Preliminary evidence suggests that the active agent(s) is a peptide with a molecular weight between 1000 and 1400 daltons.     

The inhibition by strongly acidic polypeptides of the repair of double-stranded DNA breaks induced by the gamma irradiation of Chinese hamster fibroblast cells]

The ability of polypeptides consisted of aspartic and glutamic acids to inhibit the repair and to promote the formation of unrepaired double-strand DNA breaks and chromosomal aberrations in gamma-ray induced Chinese hamster cells was shown. A complete inhibition of the double-strand DNA breaks repair was observed at the concentrations of 20 mu M/l (polyglutamic acid with molecular weight 2000-15,000 daltons) and 100 mu M/l (aspartylglutamic acid with molecular weight 1500-4500 daltons). Both polypeptides were low toxic at the given concentrations.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.     

Competition between landmarks in spatial learning: the role of proximity to the goal

In two experiments, rats were trained to find a hidden platform in a Morris pool in the presence of two landmarks. Landmark B was present on all training trials, on half the trials accompanied by landmark A, on the remainder by landmark C. For rats in Group Bn, B was near the location of the platform; for those in Group Bf, B was far from the platform. Group Bn performed better than Group Bf on test trials to B alone, but significantly worse on test trials to a new configuration formed by A and C. Thus, the spatial proximity of B to the platform affected not only how well it could be used to locate the platform, but also its ability to prevent learning about other landmarks.     

Isolation and Characterization of Two Basic Internal Proteins from the T-Even Bacteriophages

Two species of basic internal proteins were found in osmotic shock supernatant solutions of bacteriophages T4B, T4D, T2H, T2L, and T6. The major species of protein isolated had a molecular weight of approximately 21,000 daltons, whereas the minor protein molecular weight was near 9,500 daltons. The two protein species exhibited unique isoelectric points and amino acid compositions. The 21,000-dalton protein of T2L showed major electrophoretic and compositional differences from the other 21,000-dalton proteins isolated. Similarities between the 21,000-dalton proteins and phage lysozyme are discussed.     

Mechanism of Inactivation of Haemophilus influenzae Transforming Deoxyribonucleic Acid by Sonic Radiation

Transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae was exposed to sonic radiation of various durations. Reductions in transforming ability of the DNA, cellular DNA uptake, and integration into the genome, and single- and double-stranded molecular weights of the transforming DNA were measured and compared. We conclude that (i) sonic radiation causes DNA strand breaks (almost always double-strand breaks with relatively few alkaline-labile bonds), the number increasing with exposure until the double-stranded molecular weight is reduced to less than 10(6) daltons; and (ii) since transformation is reduced about as much as integration and much more than uptake, inactivation of transforming DNA by sonic radiation appears to be caused mostly by failure of Haemophilus cells to integrate the transforming DNA that is taken into the cells. These results are similar to those for inactivation by X radiation but differ from those for ultraviolet radiation. A strand break caused by sonic radiation, however, does not necessarily inactivate the transforming DNA, whereas in the case of ionizing radiation it may. The results may be fit by the model proposed by Cato and Guild. From our data and the equation of Lacks, the minimum active site of DNA necessary for transformation and the frequency of exchanges between donor and recipient strands upon integration of transforming DNA were estimated as 0.35 x 10(6) to 0.7 x 10(6) daltons and 0.15 to 0.4 switches per 10(6) daltons, respectively.