Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

In Vitro Cellular &; Developmental Biology - Plant -     

beta-Internexin, a ubiquitous intermediate filament-associated protein

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Specificities of human, rat and E. coli O6-methylguanine-DNA methyltransferases towards the repair of O6-methyl and O6-ethylguanine in DNA.

The behaviour of highly purified bacterial expressed rat O6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (km6G/ke6G = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O6-methyl and O6-ethylguanine) is similar to that of E. coli 39kD Ada protein (km6G/ke6G = 1.6). However, the human MGMT is very different (km6G/ke6G = 163). The preferential repair of O6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine. The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed. In addition the preferential repair of O6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O6-ethylguanine. This is in contrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.