Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

Isolation and structural studies on a galactofuranosyl-containing glycopeptide fromAscobolus furfuraceus

A galactofuranosyl-containing glycopeptide has been isolated from mycelium ofAscobolus furfuraceus by extraction with water. The glycoconjugate was purified by DEAE-cellulose chromatography followed by gel filtration. A molecular weight of about 20 000 was determined by the latter method using standard dextrans. Neutral sugars accounted for 94.5% of the glycopeptide and were characterized as mannose, galactose, and glucose. Glucosamine was estimated colorimetrically (1.8%). The molar ratio of Man:Gal:Glc:GlcNH₂ was 68:32:16:2. A trace amount of total phosphorus (0.2%) was found. The predominant amino acids were threonine and serine. The peptide moiety was labeled with [¹⁴C]formaldehyde and the elution of radioactivity was coincident with sugar on gel filtration in the presence of sodium dodecyl sulfate. The peak of radioactivity was retarded on release of galactose by mild acid hydrolysis. These results confirm the sugar-peptide linkage.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

Desangeloylshairidin,X-ray structure determination of a sesquiterpene lactone from Guillonea scabra

The structure and absolute configuration of desangeloylshairidin, a guaianolide isolated from Guillonea scabra, have been established by X-ray diffraction analysis. No conformational change was observed in its seven-membered ring between the crystal and deuterochloroform solution states.     

Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells

A comprehensive analysis of human alloimmune cytotoxic T lymphocytes (CTLs) specific for the HLA-A2 antigen identified 11% of HLA-A2 positive cells as outliers. In total, 11 unrelated serologically indistinguishable, but distinguishable by cell-mediated lympholysis (CML) HLA-A2 positive outlier cells were identified. The outlier cells could be subdivided in two subgroups according to reactivity patterns obtained with CTLs directed against the HLA-A2 antigen of outlier cells and their inhibitory capacity in specific competitive inhibition experiments. Thus, the serologically defined HLA-A2 specificity can be divided into at least three subtypes using CTLs specific for the HLA-A2 antigen. Moreover, CTLs specific for an HLA-A2 subtype could be induced when responder cells expressed a different HLA-A2 subtype antigen. On the basis of several family studies, we conclude that the subtype HLA-A2 antigens are inherited in a codominant way.