Mr Range Research Articles

Mouse lipocalin as an enhancer of spermatozoa motility.

The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50,000-106,000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique.

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.

Lectin isolation and detection of N-glycoproteins bearing sialic acid and L-fucose residues in human colorectal mucosa and in adenocarcinoma biopsies

A method to improve the reactivity to specific lectins of N-glycoproteins isolated from human colorectal mucosa and from adenocarcinoma biopsies was developed using a combination of techniques. Total protein extracts were subjected to affinity chromatography, using the immobilised lectin Concanavalin A coupled to Sepharose, by fast performance liquid chromatography (FPLC). N-glycoprotein enriched fractions were resolved by SDS-PAGE, transferred to PVDF membranes and incubated with various lectins. Digoxigenin-conjugated SNA I and MAA lectins were used to detect sialic acid residues. Biotin-conjugated UEA I lectin was used to detect L-fucose residues. By this method, lectin-binding N-glycoproteins were found in a broad relative molecular mass (Mr) range (from 47 to 205 kDa). No tissue-specific N-glycoproteins were observed when human colorectal mucosa and adenocarcinoma samples were compared.

The Spatial Distribution and Kinematics of Stellar Populations in E+A Galaxies

We use long-slit spectroscopic observations of the sample of E+A galaxies described by Zabludoff et al. to constrain the nature of the progenitors and remnants of the E+A phase of galaxy evolution. We measure spatially resolved kinematic properties of the young (1 Gyr) and old (few Gyr) stellar populations. The young stellar populations are more centrally concentrated than the older populations, but they are not confined to the galaxy core (radius 1 kpc). The kinematics of the old stellar population place 16 of 20 of our E+As on a trend parallel to the Faber-Jackson relation that is offset by ~0.6 mag in R. Eighteen of 20 E+As have v/σ < 1. As the young stars in these systems evolve, the luminosity offset will disappear, and the remnants will be pressure-supported systems that lie on the Faber-Jackson relation. Although Zabludoff et al. spectroscopically selected the most extreme E+A galaxies in the local volume, the sample is kinematically diverse: velocity dispersions range from 30 km s-1 to ~200 km s-1 over a luminosity range of MR = -19 to -22 + 5 log h. Combining these results with an estimate of the number of galaxies that experience an E+A phase, we conclude that the E+A phase of galaxy evolution is important in the development of a large fraction of spheroid-dominated galaxies over a wide range of luminosities and masses. Our kinematic observations, together with evidence that E+As have recently evolved from a vigorous star-forming phase to a quiescent phase (e.g., Couch & Sharples; Caldwell et al.) and that many have tidal features consistent with disklike progenitors (Zabludoff et al.), indicate that these galaxies are undergoing a transformation from gas-rich, star-forming, rotationally supported, disk-dominated galaxies into gas-poor, quiescent, pressure-supported, spheroid-dominated galaxies.

Changes in the photosynthetic apparatus of diatoms in response to low and high light intensities.

The centric diatom Cyclotella cryptica and two strains of the pennate diatom Phaeodactylum tricornutum were grown under low and high light intensities (300 lux and 3,000 lux) over 4-6 weeks. Growth was monitored by repetitive cell count. The culture media were replaced weekly to avoid morphological and biochemical alterations caused by nutrient depletion. The ultrastructure of the cells was examined by transmission electron microscopy. Alterations in the light-harvesting antenna systems were investigated by Western immunoblotting. Both diatoms reduced the plastid area, i.e. decreased the amount of thylakoid lamellae, under high light intensity. The thylakoids still ran in groups of three with parallel orientation within the chloroplasts. The girdle band lamellae were not affected. The amounts of storage compounds and vacuoles increased. SDS-PAGE of total cell protein followed by Western immunoblotting with antisera directed against subunits of the light-harvesting antenna systems of C. cryptica (cc-antiserum) and the cryptophyte Cryptomonas maculata (cmac-antiserum) revealed that both diatoms reduced the amount of antenna polypeptides under increased light intensity. The cc-antiserum immunodecorated two bands with relative molecular masses (Mr) of 18,000 and 22,000 in C. cryptica. Both decreased under high light conditions to 67.2 +/- 6.1%. Five to seven bands in the Mr range of 14,000-27,000 were recognized in P. tricornutum. They decreased to 83 +/- 5.3%. Furthermore, the immunolabeling pattern for both strains differed under the two light regimes. The cmac-antiserum immunodecorated two polypeptides with Mr of 24,000 and 23,000 in C. cryptica, while both strains of P. tricornutum had five polypeptides in the Mr range of 14,000-24,000 that showed some differences in staining intensities between the two strains and in response to the light intensity applied.

The genetic map position of the locus encoding the 2S albumin seed storage proteins in cultivated sunflower ( Helianthus annuus L.)

Sunflower contains two classes of seed proteins: the 11S globulins (helianthinins) and the 2S albumins. Polymorphisms have been detected within both classes and therefore they have been proposed as genetic markers. The objectives of the present study were to identify sunflower seed protein polymorphisms and locate them on an RFLP linkage map. Seed proteins of 68 inbred lines and four Argentinian open-pollinated populations were studied by SDS-PAGE. The helianthinins were separated into ten bands arranged in fourgroups of Mr of 22.5, 25.5, 32 and 38 kDa and all lines showed identical banding patterns. The albumins were separated into eight to tenbands in the Mr range 10 and 18 kDa. The lines showed either a band of Mr of 14.5 or of 15.5 kDa. HAR1, HAR2, ZENB8 and ZENB12 had the 15.5 kDa, whereas the remaining lines had the 14.5 kDa. The 14.5 and 15.5 kDa polypeptides were also segregating in the Impira INTA population. One hundred and fifty F2:3 families derived from ZENB8 × HA89 were used as the mapping population. The electrophoretic patterns of the families were either those of the two parental lines or presented both the 14.5 and 15.5 kDa polypeptides. No family showed absence of both bands, suggesting that they are allelic variants. The 2S albumin locus was located in the interval between the markers ZVG0051 and ZVG0052, at approximately 9 cM from ZVG0051 and at 6 cM from ZVG0052, on linkage group 11 of the public sunflower RFLP map.

Separation of Lipooligosaccharides by Linear Gradient Gel Electrophoresis

Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria. These glycolipid antigens are in the Mr range of 3–7 kDa, and SDS/PAGE has been used as an analytical tool. Although we are able to separate relatively higher Mr LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations. In the present study, we selected PID2 LOS consisting of six LOS components of 3.0–5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller Mr LOS components but also to retain resolving capability of higher LOS components. We found that mini-PAGE with stepwise and linear gradient gels (glycine–SDS) resolved smaller Mr LOS components. Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0–5.1 kDa were separated as tight and even bands. Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels. Our study also showed that preformed tricine–SDS (TSDS) minigels such as 16.5 and 10–20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels. By using glycine–SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller Mr LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past.

Effect of magnetic field and irradiation on the electrical properties and structure of the Tl-based ceramic superconductors

The voltage-current (V-I) characteristics, critical temperatures and the critical currents of Tl2Ba2CaCu2O8 (Tl-2212) and Tl2Ba2Ca2Cu3O10 (Tl-2223) ceramic superconductors were studied in magnetic fields up to 80 mT after several irradiations up to 100 MR. The effect of irradiation on the structure of these materials has also been studied. The normal state resistance increases by 20-30% with irradiation, most of the changes taking place in low doses up to 10 MR. The critical temperatures of Tl-2212 and Tl-2223 decrease by about 8 K up to 30 MR and approach the saturation values of 101 K and 113 K, respectively, with further increase of dose. For Tl-2212 in the 96-98 K temperature range the V-I behaviours become nearly ohmic under a dose of about 30 MR or in an external magnetic field of 20-30 mT. In the same temperature range the critical current decreases rapidly in low doses up to 10-25 MR; above that it decreases only slightly. For both materials, the positions and widths of the x-ray diffraction peaks almost remain the same but the peak intensities decrease by 10-40% with irradiation, the rate of decrease being higher in the 0-30 MR range. These observations indicate that drastic changes in the lattice do not take place with irradiation but substantial electronic excitations and point defects may be generated, at higher rate in low doses, to affect the concentration and arrangements of the carriers and the links between the grains.

Feasibility of NDT of Ferromagnetic Steel Structures Based on Measurements of Residual Magnetization as a Function of Elastic Stress

Algorithms for estimating elastic stress in ferromagnetic steel structures are based on measurements of the residual magnetization M r as a function of tensile and compressive stress σ0 after quenching and tempering at different temperatures T tem. The suggested testing technique is highly sensitive because the range of M r (σ0) is fairly wide (for the interval 0.6σy ≥ σ0 ≥ –0.6σy, where σy is the yield limit, the range is 600–800 kA/m). The necessary condition for using this testing technique is the availability of calibration curves for the selected testing algorithm measured on samples of steels to be tested (i.e., of the same sort of steel tempered at the same temperature). All the testing algorithms suggested in the paper enable one to determine both the magnitude and sign of the stress in the material.

Refolding and purification of a urokinase plasminogen activator fragment by chromatography

A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of Mr 10 000–1 500 000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA.

Efficacy and feasibility of breast shielding during abdominal fluoroscopic examinations

The purpose was to measure radiation exposure to the breasts during abdominal fluoroscopic examinations and evaluate the efficacy of breast shielding with a leaded vest. Sixty-six women underwent routine abdominal fluoroscopic examinations. During the examinations one breast was covered with a leaded shield. Radiation doses to both breasts were measured with a thermoluminescent dosimeter. The amount of radiation at the skin of the shielded breast was then compared with that at the skin of the nonshielded breast. Radiation exposure to the breasts varied substantially with the type of examination being performed and with the individual patient. The average radiation level at the skin of the unshielded breast was 119 mR (range, 0-6,320 mR), compared with 59.6 mR (range, 0-1,640 mR) at the shielded breast. The average reduction in radiation exposure was 50% with shielding. Although the average level of radiation exposure to the breast during abdominal fluoroscopic examinations is generally low, use of a leaded vest can further reduce radiation to the breast for different types of examinations.

Guinea pig serum contains a specific high affinity growth hormone-binding protein with novel ligand specificity.

Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current studies, however, have identified and characterized the presence of GH-binding activity in guinea pig serum using gel chromatography to separate bound and free hormone. The detection of GH-binding activity is critically reliant on the type of radioligand used to measure binding. Clear identification of GH-binding activity was demonstrated with [125I]ovine GH (oGH), but specific binding could not be measured with [125I]human GH. The novel specificity was also shared by guinea pig liver membrane GH receptor (GHR) and cytosol GHBP, suggesting structural similarity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 microl serum produced 16.03 +/- 0.5% specific binding; mean +/- SEM; n = 11) and incubation time (equilibrium was reached by approximately 6 h at 21 C) and was completely reversible (t(1/2), approximately 2 h). Scatchard analysis revealed linear plots with an affinity constant (Ka) of 0.59 +/- 0.09 x 10(9) M(-1) and a capacity of 23,181 +/- 4,474 fmol/ml serum. Similar association constants were obtained for liver membrane GHR (0.79 +/- 0.22 x 10(9) M(-1)) and cytosol GHBPs (0.99 +/- 0.15 x 10(9) M(-1)), but the capacity, when expressed as femtomoles per g tissue, was significantly increased (4-fold) in cytosol (4,303 +/- 505) over that in membranes (1,071 +/- 257). There was no sex difference in Ka or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity was very low to undetectable in pregnant guinea pig serum. Characterization of the native structure of guinea pig GHBPs has indicated the presence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large Mr range (approximately 70-350 kDa) the major form of the circulating GHBP identified by gel chromatography had an apparent native Mr of 150-170 kDa. Partially purified GHBP (approximate Mr, 170 kDa) was covalently cross-linked to [125I]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP complexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated noncovalently with a non-GH-binding protein. "Pore limit" native PAGE (cathodic and anodic) revealed the presence of specific GHBPs of 363, 158, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-specific mAb 4.3. Interestingly, although GH binding was undetectable in pregnant guinea pig serum, Western immunoblot analysis with mAb 263 demonstrated the presence of a major immunoreactive GHBP band of 105 kDa in addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no evidence to support the presence of a hydrophilic tail sequence homologous to that in the rat GHBP. These studies have identified in guinea pig serum GHBPs that exhibit novel ligand specificity, structural heterogeneity, and an immunological relationship to the rat liver membrane GHR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.

The Light Harvesting System of the Diatom Cyclotella cryptica. Isolation and Characterization of the Main Light Harvesting Complex and Evidence for the Existence of Minor Pigment Proteins*

Abstract:The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N‐ter‐mini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)+ RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less β‐carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five‐fold relative to chlorophyll a under high light conditions. Western‐immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.

Electrophoretic seed globulin patterns in some New World Lupinus species

Comparative SDS-PAGE analysis of seed globulins covered 74 accessions representing 19 New World Lupinus species, 18 North American lupins and one South American species: L. mutabilis. The species investigated showed major, well-defined polypeptide bands in the approximate Mr range of 40–75 kDa. In general, intraspecific variation blurred interspecific differences; L. mutabilis could not be distinguished from the North American lupins. Of the taxa investigated, only L. subcarnosus and L. succulentus displayed some species-specific features. The American species examined showed different seed globulin patterns from those reported earlier for the smooth-seeded and for the rough-seeded Old World lupins.

Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products.

This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface. The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus. Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT. Western blot (immunoblot) analysis of P. gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000. Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P. gingivalis DNA. These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P. gingivalis in periodontal disease.

A DEVELOPMENTAL STUDY OF PROTEIN PHOSPHORYLATING SYSTEMS STIMULATED BY PHORBOL DIBUTYRATE IN MICRO-SLICES OF RAT BRAIN

Age-dependent changes in the activity of protein phosphorylating systems stimulated by phorbol dibutyrate (Mr range 40–80 kDa) were studied in micro-slices from rat brain labelled with [32P]orthophosphate. In adult animals phorbol stimulated the phosphorylation of the known substrates of protein kinase C (B-50/GAP-43 and MARCKS) and 3 unknown polypeptides: a basic molecule of 74 kDa (pp74B) and acidic molecules of 60 kDa (pp60A) and 42 kDa (pp42C). In neonatal animals the labelling of MARCKS was relatively very high as previously reported. Labelling of pp60A was present at birth, but increased during development; labelling of pp74B was only detected after days 8–10. The activity of the system labelling pp42C was very high during the first 2 weeks postnatal and then declined rapidly. The labelling of pp74B was considerably higher in slices from the cerebral cortex compared with the hippocampus; no regional differences in the labelling of pp60A were observed. Compared to B-50/GAP-43 and MARCKS, stimulation of phosphorylation of pp74B and pp60A required a higher concentration of phorbol. The substrates of all the phorbol-responsive systems, with the exception of pp74B, were soluble in 40% acetic acid. The possible identity of pp60A, pp74B and pp42C is discussed. Copyright © 1996 Elsevier Science Ltd.

Physico-Chemical Properties and the Structure of Dermatan Sulfate Fractions Purified from Plasma after Oral Administration in Healthy Human Volunteers

Dermatan sulfate (DS) was administered by oral route in healthy human volunteers. The structure, physico-chemical properties and biological activity of DS purified from human plasma after oral administration were studied and compared with those of native DS. DS extracted and purified from pig mucosa has a relative molecular mass (Mr) of about 23,100 and is composed of about 10% nonsulfated disaccharide, 80% monosulfated disaccharides and about 10% disulfated disaccharides, with a sulfate to carboxyl ratio of 1.00 and a heparin cofactor II (HCII) activity of about 160 units/mg. This native polysaccharide is composed of about 94% iduronic acid. One gram of native DS was orally administered to five healthy human volunteers, and 50 ml of blood were collected after 4 h. DS possibly present in plasma after oral administration was extracted and purified. About 130 +/- 42 micrograms of DS per 50 ml of blood were detected by agarose-gel electrophoresis and DMB assay. This DS shows a broad Mr range. After oral absorption, substantial amounts of species with a Mr of about 7,500 are detected in blood but chains with Mr ranging from 7,500 to 20,000 are also found. Moreover, some very low-Mr species are detected, with a prevalence of disaccharides. After oral absorption, DS is sulfated above all in position 4 of the N-acetyl-galactosamine (60%), with a sulfate to carboxyl ratio of about 0.64, demonstrating that DS is desulfated during or after oral absorption by about 30-40%, A small amount of disulfated disaccharide (in particular 2,4-disulfated, 1.4%) is preserved from catabolic processes, as DS extracted from human plasma is able to inhibit thrombin activity mediated by HCII (about 16 U/mg).

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (Mr), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the Mr of unbranched cell-wall polymers in the Mr range of 106–107 Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.

Biochemical variability between venoms from different honey-bee (Apis mellifera) races

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences. 2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former. 3. The polypeptides with Mr in the range from 100,000 to 7500 Da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with Mr range from 4100 to 2000 Da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.

The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host.