Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.

Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.

Anti-Infectivity Activity of Human Salivary Secretions toward Human Immunodeficiency Virus

The purpose of this investigation was to adapt an MT-2 cell syncytium-forming assay for measuring anti-infectivity activity of salivary secretions toward HIV and to determine the distribution of this activity in a population of healthy adult subjects. Whole saliva samples were collected from 27 volunteers, who reported that they did not belong to any group at high risk for HIV infection, and tested for anti-infectivity activity using the syncytium-forming assay. Nine of these subjects were subsequently retested on one or more occasions to assess the variability in appearance of this activity. Parotid and extraparotid salivas of six subjects were also tested. Samples were frozen immediately after collection and submitted in blinded fashion for quantitation of their anti-HIV activity using a syncytia-forming MT-2 cell assay or the p24 antigen ELISA. Nine out of the 27 subjects showed detectable anti-HIV infectivity activity. One parotid sample and one extraparotid sample out of four from subjects with positive whole salivas were positive and none of the parotid or extraparotid samples from two subjects with negative whole salivas were positive. The inhibitory activity ranged from 0.5 to 1 log 10 TCID50 /ml and could not be correlated with total protein content in saliva or any specific electrophoretic component. Filtration of the saliva through an Amicon 10 filter before incubation with the virus abolished the activity. Similar studies using two other biological fluids, urine and cerebrospinal fluid, revealed no anti-HIV infectivity activity. These findings confirm the presence in saliva of inhibitory activity directed toward HIV.

Dental Enamel Matrix — Isolation and Partial Sequence of a Peptide Component

Chromatography of demineralized bovine fetal enamel matrix proteins on 'Biogel P6' was shown to yield a trailing peak which contained a single electrophoretic component. This polypeptide, further purified by chromatography on Biogel P4', was found to be homogeneous by disc electrophoresis and gel isoelectric focussing. Amino acid analyses indicated this component (designated: 'E5') to be similar to some of the phosphopeptides previously described. Cyanogen bromide cleavage of E5 yielded three principal products whose amino acid compositions and N-terminal residues were investigated. A partial sequence for component E5 was proposed and comparison with previous bovine matrix isolates was made.

The acid phosphatases of bovine leucocytes, plasma and the milk of healthy and mastitic cows

SummarySome of the acid phosphatase isozymes of bovine leucocytes and plasma have been separated and partly characterized. About 80% of the phosphatase activity of leucocytes at pH 4·9 was particle-bound and about 8% was extractable with Amberlite CG-50 ion exchange resin. This extractable enzyme existed as a single electrophoretic component with a mol. wt of about 42000 and with optimum activity at pH 5·8. Km for p-nitrophenyl phosphate was 1·6 mM at pH 5·8 and 0·4 mM at pH 4·9. At pH 5·8 orthophosphate (K1 = 1·5 mM) and pyrophosphate (Ki = 4·1 mM) were competitive inhibitors. The enzyme was also strongly inhibited by F−, Al3+, IO4− and S2032−. The enzyme which was not extractable with Amberlite was very heterogeneous with respect to molecular weight. At the pH optimum (4·9), Km for p-nitrophenyl phosphate was 0·4 mM and orthophosphate (K1 = 2·3 mM) and pyrophosphate (K1 = 2·1 mM) were competitive inhibitors. Other inhibitors included F−, Al3+, Hg2+, IO4− and tartrate. The enzyme extracted from plasma by Amberlite CG-50 treatment had properties similar to that extracted from leucocytes. Normal bovine milk contained a single acid phosphatase, but milk from cows with mastitis showed 3 electrophoretic isozyme bands, one being the same as in normal milk; the 2 additional bands were of leucocyte origin.

Chain Termini of the Satellite RNA from Yeast Ribosomes

(1) When yeast cells are extracted with aqueous phenol and the aqueous phase is made 2.5 M with respect to NaCl at 0°, there is selective precipitation of about 80% of the total RNA. The 17 S and 26 S RNA from yeast ribosomes comprise a preponderant mass-fraction (ca. 90%) of this NaCl-insoluble RNA. A small amount (ca. 3%) of a rapidly migrating electrophoretic component (iRMEC) can be released by aqueous denaturation of yeast NaCl-insoluble RNA (iRNA), and because it forms a specific complex with 26 S RNA, this iRMEC component can be appropriately described as a "satellite" of 26 S RNA.(2) Following its release by aqueous denaturation of yeast NaCl-insoluble RNA, the satellite RNA has been subjected to end-group analysis, and it has been found to have a formal structure that is based on a repeating 5′-mononucleotide unit, i.e. (pN)n, where n = 160–200. On the basis of an analysis of its principal termini, the dominant form of the satellite RNA is[Formula: see text](3) In an allied study, a "rapidly labelled" fraction of yeast NaCl-insoluble RNA has been subjected to aqueous denaturation and end-group analysis.

Wheat-Embryo Ribonucleates. I. Subcellular Localization of a Satellite Polyribonucleotide

(1) When wheat embryos are extracted with aqueous phenol and the aqueous phase is made 2.5 M with respect to NaCl at 0 °C, there is selective precipitation of about 80% of the total RNA. The 18 S and 26 S RNA species from the wheat-embryo ribosomes comprise a preponderant mass fraction (ca. 80%) of this NaCl-insoluble RNA (iRNA). A small amount of a rapidly migrating electrophoretic component (iRMEC) can be released by aqueous denaturation of wheat-embryo NaCl-insoluble RNA and because it is specifically complexed with 26 S RNA, the iRMEC component has been termed a "satellite" of 26 S RNA.(2) The wheat-embryo satellite RNA has been shown to be present in the microsomal fraction recovered from cell-free homogenates of wheat embryos.(3) The wheat-embryo satellite RNA has been shown to be differentially localized in the large subunit of wheat-embryo ribosomes where it presumably exists as part of the same intermolecular 26 S RNA complex that can be isolated by directly extracting the whole embryos with aqueous phenol.(4) During preparation of the ribosomal subunits, there is substantial degradation of the component ribonucleates and the nature of this degradation is the subject of a brief discussion.

GROWTH HORMONE ACTIVITY IN MAN OF CHYMOTRYPTIC DIGESTS OF BOVINE GROWTH HORMONE

SUMMARY Bovine growth hormone (BGH) digested with chymotrypsin showed a significant retention of biological activity after the hydrolysis of six bonds and the formation of 20% non-precipitable nitrogen. Disk electrophoresis of ½ and 3 hr. digests demonstrated complete loss of the major electrophoretic component of BGH with the appearance of one or more components of greater anodal mobility. Administration of the chymotryptic digests of BGH to three hypopituitary patients resulted in an anti-insulin response, aggravation of diabetes mellitus or anabolic effects.