Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF. The substitution of Pro1743, the residue immediately adjacent to the Arg-Gly-Asp sequence on the amino-terminal side, with Phe resulted in a peptide that failed to interact with either antibody. Thus, Pro1743 is important for maintaining a peptide conformation recognized by two antibodies specific for the GP IIb-IIIa-binding domain of vWF. Other residues important for optimal peptide reactivity with the polyclonal antibody were Ser1742, Arg1744, and Gly1745, whereas Gly1741, Gly1745, and Asp1746, but not Arg1744, were important for reactivity with the monoclonal antibody. The epitopes of both antibodies, therefore, included at least 2 of the residues in the sequence Arg-Gly-Asp considered the common cell-binding site of adhesive molecules that interact with GP IIb-IIIa. Nevertheless, both antibodies reacted only with vWF. These studies demonstrate that peptide-specific antibodies, unlike the promiscuous GP IIb-IIIa receptor, can recognize distinctive structural characteristics of the cell-binding domain of adhesive molecules imposed by residues adjacent to the sequence Arg-Gly-Asp.     

The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen

The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus. The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAGQAKKKKLD, and the sequence AVKRPAATKKAGQAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism.     

Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.     

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.     

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.     

Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.

We have isolated a second gene (MLS1), which in addition to DAL7, encodes malate synthase from S. cerevisiae. Expression of the two genes is specific for their physiological roles in carbon and nitrogen metabolism. Expression of MLS1, which participates in the utilization of non-fermentable carbon sources, is sensitive to carbon catabolite repression, but nearly insensitive to nitrogen catabolite repression. DAL7, which participates in catabolism of the nitrogenous compound allantoin, is insensitive to carbon catabolite repression, but highly sensitive to nitrogen catabolite repression. Results obtained with null mutations in these genes suggest that S. cerevisiae contains at least one and perhaps two additional malate synthase genes.     

A second gene (qutH) within the Aspergillus nidulans-quinic-acid utilisation gene cluster encodes a protein with a putative zinc-cluster motif.

A sequence of 3299 nt, contiguous with the previously sequenced quinate permease-encoding (qutD) gene and encompassing the dehydroshikimate dehydratase-encoding (qutC) gene, has been determined. Northern-blot analysis detected (i) a quinate-inducible mRNA of the expected size for the qutC gene, and (ii) a quinate-inducible mRNA of 1.45 kb divergently transcribed away from qutC towards qutD. Computer-aided sequence analysis identified an ORF of 1047 nt corresponding to the qutC gene encoding dehydroshikimate dehydratase. In addition, a genetically uncharacterized 1188-nt gene, designated qutH and containing a putative intron of 61 nt, was identified between qutC and qutD. The inferred protein sequence encoded by qutH contains a putative 'zinc cluster' motif and has a low (16%) but significant similarity with the DNA-directed DNA polymerase of hepatitis B virus. The results are interpreted as being consistent with the view that the qutH gene encodes a DNA-binding protein, possibly involved in the regulation of genes essential for the utilisation of protocatechuic acid.