Low Molecular Weight Basic Proteins Research Articles

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice

Objectives Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Methods Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Results Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. Conclusion The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis.

Biochemical characterization of buffalo (Bubalus bubalis) milk lysozyme.

Lysozyme, a low-molecular weight basic protein, is an important component of the antibacterial system in milk. Lysozyme activity is higher in buffalo milk (60±3·9×10−3 units/ml) than in bovine milk (29·1±1·5×10−3 units/ml). Buffalo colostrum contains five-times more lysozyme activity than mature milk (Priyadarshini & Kansal, 2002a). Lysozyme activity in buffalo milk is not influenced by the parity of animal or stage of lactation, but it increases during extreme weather (winter and summer). Lysozyme in buffalo milk is more stable than in cow milk during storage and heat treatment. A sharp increase in milk lysozyme has been observed in buffaloes with sub-clinical mastitis (Priyadarshini & Kansal, 2002a).

A Cold‐Regulated Protein from Bermudagrass Crowns Is a Chitinase

Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography of in vivo radiolabeled proteins has revealed that protein synthesis in crowns of ‘Midiron’ and ‘Tifgreen’ bermudagrasses (Cynodon dactylon L. Pers. × C. transvaalensis Burtt‐Davy) is altered in association with cold acclimation (CA). Increased synthesis of some proteins in association with CA in Midiron crowns correlates with the greater freezing tolerance of Midiron compared with Tifgreen. This study was undertaken to (i) characterize further alterations in protein synthesis in Midiron and Tifgreen crowns utilizing analytical isoelectric focusing (IEF)/SDS‐PAGE [two‐dimensional (2D‐) PAGE], and (ii) identify cold‐regulated (COR) proteins, proteins encoded by genes persistently upregulated during CA, through sequence analysis of peptides recovered after micropreparative 2D‐PAGE and protease digestion. Plants were grown for 26 d in controlled‐environment chambers under acclimating [8/2°C (day/night) cycles with 10 h photoperiods] or nonacclimating [28/24°C] conditions. Proteins synthesized by isolated crowns were radiolabeled in vivo for 16 h with 35S‐methionine and 35S‐cysteine. Among several groups of COR proteins synthesized in Midiron and Tifgreen crowns, low molecular weight (about 20–28 kilodalton) basic (LB) COR proteins were synthesized in greater amounts and numbers in Midiron than Tifgreen crowns. Among the LB COR proteins, a 27‐kD basic COR protein was synthesized prominently in crowns of both Midiron and Tifgreen plants. This protein from Midiron crowns was designated COR27 and identified as a chitinase (E.C. 3.2.1.14) through protein sequencing.

A Haploid Expressed Gene Cluster Exists as a Single Chromatin Domain in Human Sperm

Mammalian spermiogenesis is marked by the initial disruption of the nuclear-histone-DNA complex by the transition proteins for ultimate replacement with protamines. The genes for three of these low molecular weight basic nuclear proteins exist as a single linear array of PRM1, PRM2, and TNP2 on human chromosome 16p13.2. To begin to address the mechanism governing their transcriptional potentiation, a region of approximately 40 kilo-bases of the human genome encompassing these genes was introduced into the germ line of mice. Fluorescence in situ hybridization and Southern analysis showed that this segment of the human genome integrated into independent chromosomal sites while maintaining its fidelity. Transcript analysis demonstrated that the expression of the endogenous mouse protamine Prm1 and Prm2 genes as well as the mouse transition protein Tnp2 gene were expressed along with their human transgene counterparts. The pattern of expression of these transgenic human genes within this multigenic cluster faithfully represented that observed in vivo. In addition, all members of this transgenic gene cluster were expressed in proportions similar to those in human testis. Copy number-dependent and position-independent expression of the transgenic construct demonstrated that the corresponding biological locus was contained within this segment of the human genome. Furthermore, DNase I sensitivity established that in sperm the human PRM1-->PRM2-->TNP2 genic domain was contained as an approximately 28.5-kilobase contiguous segment bounded by an array of nuclear matrix associated topoisomerase II consensus sites. This is the first description of a multigenic male gamete-specific domain as a fundamental gene regulatory unit. A model of haploid-specific gene determination is presented.

Coating porous silica by in situ polymerization of peo macromonomers for size exclusion separation of proteins

A coating method using comb-like polymers with poly(ethylene oxide) (PEO) grafts is described and studied in order to use porous silica as phase in size exclusion chromatography (SEC) of proteins. The coating is carried out by in situ polymerization and crosslinking of methacrylates of PEO with OH end groups (macromonomers) after deposition from a solution. A series of phases was prepared using two different graft lengths and two different crosslinking agents in different proportions. The efficiency of the coatings to mask the silanol groups of the matrix is characterized by pH metric titration and the best coating conditions are deduced. From calibration with PEO standards and with proteins, the SEC behavior of each phase is analyzed from column experiments and compared to results of pH metric measurements. A close interdependence between the two methods is observed. The optimized phases exhibit very good performance in SEC, except with low molecular weight basic proteins which interact with some residual unmasked silanol groups. The presence of these groups is attributed to the intrinsic porosity of the polymeric layer which increases with the length of the grafts.

Purification and characterization of a low molecular weight basic protein from marine turtle egg white

The egg white of marine turtle ( Caretta caretta Linn.) and one species of tortoise ( Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g −1 in 0.2 m NaCl. It has a Stokes radius of 12.6 Å and a diffusion coefficient of 16.50 × 10 −7 cm 2 s −1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% β-pleated sheet and little or no α-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine, and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basic protein is partially resistant to the action of DNase.

Isolation and partial characterization of a human alveolar macrophage-derived neutrophil-activating factor.

Human alveolar macrophages (AM) were obtained from eight normal volunteers using fiberoptic bronchoscopic lavage to explore potential interrelationships among leukocytes in pulmonary defense against infection. AM placed in monolayer tissue cultures released material into culture supernatants with the capacity to enhance the bactericidal capacity of human neutrophils. Neutrophils preexposed to supernatants killed Pseudomonas aeruginosa from 70 to 90% more efficiently than control cells (P less than 0.02). AM culture supernatants contained this material by 4 h of incubation, and in vitro stimulation of AM cultures with heat-killed P. aeruginosa further increased its production. Gel filtration of AM culture supernatants with a G-50 Sephadex column allowed isolation of a 6,000-D neutrophil-activating factor (NAF) that was resistant to heat (56 degrees C, 30 min). The isoelectric point of NAF, as determined by chromatofocusing, was approximately 7.6. Enzyme digestion of NAF specimens, prepared sequentially by gel filtration and chromatofocusing, demonstrated 50-70% loss of activity after incubations with trypsin, chymotrypsin, and neuraminidase. NAF was only minimally chemotactic and eluted from Sephadex G-50 with particles of a different molecular size than those of AM-derived chemotactic factors (i.e., approximately 10,000 D and less than 500 D). Preincubation of neutrophils with NAF resulted in greater release of superoxide anion upon their subsequent stimulation by either bacterial phagocytosis or by phorbol myristate acetate, as compared with control neutrophils stimulated in a like manner. These studies indicate that human AM secrete a heat-stable, low molecular weight basic protein, with the capacity to enhance oxidative microbicidal activity of neutrophils.

RNase a effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25°C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment of HeLa cell cytosol at 2°C results in receptor complexes which sediment at 6.9S in low salt gradients. RNase A treatment at 25°C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2°C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25°C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25°C, but not at 2°C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating.Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25°C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2°C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25°C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A.These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.

Extraction purification and characterization of the sperm protamines of the dog-fish Scylliorhinus caniculus

Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with othe sperm-specific protamines previously described.

Interaction of ribonuclease A with estrogen receptor from rat mammary tumor MTW9.

Incubation of crude estrogen receptor preparations from mammary tumor cytosol with RNase A increases the sedimentation coefficient of the receptor from 9.7 S to 10.4 S. The effect is not obtained with other low molecular weight basic proteins (lysozyme, cytochrome c, or histone H2B). Nonenzymically active RNase A derivatives such as performic acid oxidized RNase A, fully reductively methylated RNase A, carboxymethyl-His-119-RNase A, and RNase S-protein were ineffective. RNase T1, an acidic endoribonuclease, was also without effect. However, enzymically active RNase S', prepared from a mixture of RNase S-protein and S-peptide, shifted the sedimentation to 10.4 S. The increased sedimentation is not accompanied by a change in the Stokes radius of the receptor (74 A) or buoyant density in metrizamide (1.24 g/ml). The effect of RNase A on the sedimentation of the receptor can be reversed by subsequent incubation with human placental RNase inhibitor or with rabbit anti-RNase A antibodies. Direct interaction was shown by chromatography of the receptor on RNase A Sepharose. Thus, the shift in sedimentation results from binding of RNase A to the receptor and, although this requires that the enzyme active site be available, enzymic activity is not responsible for the effect. The interaction of RNase A with the receptor occurs at low ionic strength; it does not occur at elevated ionic strength or after activation of the receptor by precipitation with ammonium sulfate.

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD 50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and trytophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some toxins derived from the same venom 5. It is the first chemically characterized chactoid toxin.

Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses.

Murine leukemia viruses contain a low molecular weight basic protein, designated p10, which binds to single-stranded nucleic acids. The complete amino acid sequence of p10 from the Rauscher strain of virus has been determined. The partial amino acid sequences of p10s from Moloney, Friend, AKR, Gross, radiation leukemia, and BALB/2 viral strains have also been determined using microsequencing techniques. Rauscher p10 is composed of 56 amino acid residues; the other p10s are similar in size but differ from Rauscher by a few conservative amino acid substitutions. The structure of Rauscher p10 was compared to the structure of a functionally homologous protein from Rous avian sarcoma virus. The comparison revealed regions of amino acid sequence homologies which indicate a phylogenetic relationship between the murine and avian viral strains. The analyses revealed a periodic placement of three Cys residues and a Gly-His sequence. A structure involving these residues is found once in the murine protein and twice in the avian protein. A similar structure is seen in the single stranded nucleic acid binding protein of bacteriophage T4. However, in the latter case, the order of amino acid residues is inverted.

Analytical techniques for cell fractions: XXVI. A two-dimensional electrophoretic analysis of basic proteins using phosphatidyl choline/urea solubilization

The ISO-DALT system of two-dimensional electrophoresis allows high-resolution separations of proteins and protein subunits. However, the conventional isoelectric focusing employed in this system does not give satisfactory resolution of the more basic proteins, such as histones. The BASO-DALT system was designed to obtain improved resolution of these basic proteins in the first dimension. In this system, phosphatidyl choline is used as the solubilization agent, and allows resolution of many low molecular weight basic proteins that were not seem with more conventional detergents. Using the BASO-DALT system, Novikoff hepatoma chromosomal proteins have been analyzed, and the five histones identified.

Biochemical and pathological studies of myelin in hexachlorophene intoxication

Adult female rats were fed a diet containing 500 ppm hexachlorophene (HCP). Morphological study of brains from these animals showed vacuolation of the myelin sheaths due to separation of myelin lamellae at the minor dense line. However, myelin could be isolated from the brains of these animals in normal yield. The myelin isolated from HCP-fed animals had normal lipid and protein compositions as shown by analyses of the individual lipids and by disc gel electrophoresis of the proteins. Assay of the myelin-specific enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase, showed normal specific activity in myelin obtained from HCP-fed rats. Brains of HCP-fed rats showed an increase in wet weight and a decrease in dry weight, with the chloroform-methanol insoluble fraction showing the greatest weight loss. During isolation of myelin from HCP-fed rats material was found floating over 0.32 M sucrose. This "floating fraction" contained a higher ratio of lipid to protein but the same relative proportions of the individual lipids as are found in myelin. The yield of "floating fraction" from each HCP-fed rat was less than 10 percent of the yield of myelin. Disc gel electrophoresis demonstrated the presence of the usual myelin proteins in this fraction, but with a slight increase in the relative amount of the low molecular weight basic protein. The data were compared to reports on the biochemistry of triethyltin poisoning, and it was concluded that vacuolation of myelin in HCP poisoning is probably due to increased permeability of myelin lamellae to water and electrolytes.

Purification and properties of some unique low molecular weight basic proteins degraded during germination of Bacillus megaterium spores.

Two major proteins, termed proteins A and B, and one minor species, termed protein C, have been purified to homogeneity from dilute acid extracts of dormant spores of Bacillus megaterium. These three species comprise approximately 80% of the protein in the dilute acid extracts and account for 60 to 75% of the protein degraded during spore germination. All three proteins have low molecular weights (7,000 to 10,000), high isoelectric points (greater than 9.8), alanine as the NH2-terminal amino acid, are more hydrophilic than most proteins, and all lack cysteine, cystine, and tryptophan. In addition all three proteins are extremely sensitive to a wide variety of proteolytic enzymes, much more so than "average" proteins such as serum albumin, lysozyme, and hemoglobin. These proteins also bind to both purified DNA and to a nuclear body from dormant spores. Although this binding gives little or no protection to proteins A and B from proteolysis, it does result in elevation of the melting temperature of the DNA by as much as 20degrees.

A protein toxic to crustacea from the venom of the scorpion Androctonus australis

Using column chromatography, a protein, selectively toxic to isopods (Crustacea) has been isolated and purified from the venom of the scorpion Androctonus australis. The final product is about 250 times more toxic than the crude venom. It is a low molecular weight (70 amino acids) basic protein, devoid of methionine, histidine, and phenylalanine, rich in arginine and supposed to contain five disulfide bridges. The amino acid composition of this toxin is compared to those specifically active to mammals as well as to insects derived from the same venom, and the significance of such selectivity in action is discussed.

Identification of several unique low molecular weight basic proteins in dormant spores of Bacillusmegaterium and their degradation during spore germination

Summary Approximately 20% of the total protein of Bacillus megaterium spores is degraded during the first 20 minutes of spore germination, and > 90% of the degradation can be accounted for by a fraction extracted from disrupted spores with dilute acetic acid. This fraction from dormant spores contains only two major proteins on disc gel electrophoresis at pH 4.3. These protein bands are not seen in acetic acid extracts from germinated spores, vegetative cells or early stationary phase cells, but they appear during sporulation about 1 hour prior to the appearance of refractile spores. The proteins in the acetic acid soluble fraction from dormant spores are all of low molecular weight (7–12,000) and are quite basic (pI > 9.6).

Resolutions and identification of the core deoxynucleoproteins of the simian virus 40

Low molecular weight basic core proteins of SV 40 are resolved by Tris-Acetate-SDS polyacrylamide gel electrophoresis into a minimum of four polypeptides. These are the electrophoretic counterparts of the evolutionarily conserved histones F3, F2b, F2a2, and F2a1. Host African green monkey kidney cells contain an active protease activity which readily cleaves histone F3 during nuclear isolation in hypotonic buffers.

Cytogenetics in Clinical Medicine

The purpose of this article is to provide basic information about (1) the techniques used to prepare human chromosomes, (2) the nomenclature used in identifying human chromosomes, and (3) the detection of chromosomal aberrations. Information currently accumulated about human chromosomal abnormalities has been reviewed elsewhere. 1 Chromosomes are the units of organization of deoxyribonucleic acid which code for the genetic information present in the nucleus of the cell. In addition to DNA, chromosomes contain ribonucleic acid, histone (low molecular weight basic protein), and a more complex acidic residual protein. The manner in which these four macromolecules are bound together to form a chromosome is not yet fully understood. The DNA in the chromosomes undergoes exact replication before a cell divides. For most of the cell cycle, chromosomes are in an extended state and cannot be counted or identified. Early in cell division the chromosomes begin to contract so that by

Isolation and Characterization of a Low Molecular Weight Basic Protein of Normal Human Plasma

Abstract A method was developed for the purification of a basic protein, designated as B2, derived from normal human plasma. Consecutive chromatography on carboxymethyl cellulose, diethylaminoethyl sephadex, and hydroxylapatite yielded a preparation that was homogeneous on starch gel electrophoresis. The plasma protein, B2, was characterized in terms of its major physicochemical and chemical properties. The molecular weight was determined to be approximately 9000 and the s20,w0 value 1.3 S. The calculated shape of B2 corresponds to an ellipsoid of revolution with a frictional ratio of 1.25 and an axial ratio of 4:5. The isoionic and isoelectric points of this protein were found to be at approximately pH 10.7 and 10.1, respectively. At pH 8.6 the electrophoretic mobility of this blood constituent was +1.3 x 10-5 cm2 sec-1 volt-1. B2 possesses one polypeptide chain as deduced from one amino-terminal amino acid, i.e. aspartic acid (or asparagine), and one carboxyl-terminal amino acid, i.e. glycine. It is devoid of carbohydrates and is further distinguished by the lack of histidine and tryptophan. The basic nature of this protein is due to the high amide content rather than a high content of basic amino acids. Based on optical rotatory dispersion measurements B2 has α-helical structure involving about 50% of the amino acid residues.