Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

Localization of a gene for human α-galactosidase B (=N-Acetyl-α-D-Galactosaminidase) on chromosome 22

Summary The localization of the structural gene for human -galactosidase B (=N-acetyl--galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman -galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl--galactosominidase. The results show that the structural gene for human -galactosidase B is situated on chromosome 22, and that there is no structural relationship between human -galactosidase A and human -galactosidase B.     

The complete primary structure of the T -Cell receptor genes from an alloreactive cytotoxic human T-lymphocyte clone

The complete primary structure of the cDNAs encoding the and chains of the T-lymphocyte receptor for antigen from a human alloreactive, cytotoxic T-cell clone, L17, is presented. Sequence analysis of these genes reveals that both are related to immunoglobulins and are composed of variable, diversity (at least in the case of the Ti clone), joining, and constant region sequences. Comparison of the sequence of the -chain cDNA to that of previously sequenced mouse and human cDNAs suggests the presence of human T-cell receptor D-region sequences. Southern blot analysis confirms the finding that these cDNAs represent the functional receptor genes expressed by the L17 cytotoxic T-cell clone. The availability of these full-length T-cell receptor cDNA clones from a human T-lymphocyte clone of known antigen specificity should allow an analysis of the relationship between T-cell receptor structure and function.     

Expression of ACTH-induced corticosteroid biosynthesis in newborn rat adrenocortical cells cultured in serum-free and carrier protein-free medium containing a cholesterol-α-cyclodextrin complex

Newborn rat adrenocortical cells were successfully cultured in a serum free carrier protein free medium (SPFM) by using -cyclodextrin as a cholesterol carrier and have expressed corticosteroid biosynthesis in this medium. A stable inclusion complex of cholesterol--cyclodextrin with a molar ratio of almost 1 was obtained for a 5 × 10^–5 mol/1 -cyclodextrin concentration. Cell cultures incubated with [4-¹⁴C] cholesterol--cyclodextrin in SPFM produced, under ACTH stimulation, various ¹⁴C labeled steroids with a predominance of corticosterone and 18-hydroxy-11-deoxycorticosterone. As measured by gas chromatography and mass spectrometry, the ratio between corticosteroids (21-hydroxylated steroids) and 20-reduced steroids produced in SPFM with cholesterol--cyclodextrin was equal to 1.8. This corresponds to a value of 3.6 times higher than that found in the serum free medium with cholesterol-albumin. Consequently, the chemically defined SPFM with cholesterol--cyclodextrin used in this study is more suitable for corticosteroidogenesis by adrenal cells in culture than a serum free medium with cholesterol-albumin.Abbreviations -CD -cyclodextrin - ACTH adrenocorticotropic hormone - 20-dihydroprogesterone 20-hydroxy-4-pregnene-3-one - 11-hydroxy-20-dihydroprogesterone 11, 20-dihydroxy-4-pregnene-3-one - 11-hydroxyprogesterone 11-hydroxy-4-pregnene-3,20-dione - C--CD cholesterol--cyclodextrin complex - corticosterone 11,21-dihydroxy-4-pregnene-3,20-dione - deoxycorticosterone 21-hydroxy-4-pregnene-3,20-dione - 18-hydroxy-11-deoxycorticosterone 18,21-dihydroxy-4-pregnene-3,20-dione - 18-hydroxy-20-dihydroprogesterone 18-20-dihydroxy-4-pregnene-3-one - 18-hydroxyprogesterone 18-hydroxy4-pregnene-3,20-dione - progesterone 4-pregnene-3,20-dione - SFM-S serum-free medium - SPFM serum-free protein-free medium - SSM serum supplemented medium     

Expression of the mRNA encoding truncated PPARα does not correlate with hepatic insensitivity to peroxisome proliferators

Two alternatively spliced forms of human PPAR mRNA, PPAR1 and PPAR2, have been identified. PPAR1 mRNA gives rise to an active PPAR protein while PPAR2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR1 and PPAR2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR2 mRNA abundance is approximately half that of PPAR1 mRNA; a correlation analysis of PPAR1 and PPAR2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR2/PPAR1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR2/PPAR1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPARa activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR2/PPAR1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR2/PPAR1 mRNA. These data suggest that selective PPAR2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.     

Breakage of the T cell receptor α chain locus in non malignant clones from patients with ataxia telangiectasia

Summary Patients with ataxia telangiectasia (A-T) develop specific chromosome translocations, which may confer a proliferative advantage, resulting in the appearance of large clones in the peripheral blood lymphocytes. These lymphocytes are not malignant. Using in situ hybridisation techniques we have investigated a consistent 14q11 translocation break-point observed in a t(X;14)(q28;q11) translocation clone from each of two different patients and a t(14;14)(q11;q32) clone from a third patient. In all cases the chromosome translocation involved breakage within the chain locus of the T cell receptor (TCR), between the variable and constant regions, at 14q11. Chromosome rearrangement involving breakage within TCR can therefore precede the development of malignancy. Further chromosomal rearrangement may be required in these patients, for progression to the leukaemic state.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α, β, or γ genes

To test the hypothesis that the T-cell receptor (Tcr) gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16⁺ lymphocytosis were analyzed for rearrangements and expression of the human Tcr , , and genes. Two of the clones displayed distinct rearrangements of their Tcr and genes and expressed mature Tcr , , and l RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr and gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.     

Molecular cloning of the translocation breakpoint in T-ALL 11;14 (p13;q11): genomic map of TCR alpha and delta region on chromosome 14q11 and long-range map of region 11p13

Summary Using chromosome walking techniques, overlapping lambda and cosmid clones from the T cell receptor alpha (TCR) region have been isolated; these span the entire J region and parts of the TCR delta gene. Molecular analysis of the acute childhood leukemia cells (T-ALL) 8511 revealed a rearrangement on one chromosome 14 in J 58 kb 5 of C; this does not result in production of message. The translocation was identified 90 kb 5 of C at the previously identified J² element. A probe derived from the 5 region of the translocation breakpoint hybridized to DNA from a mouse-human cell hybrid containing chromosome 11 as the only human chromosome. This probe was used to isolate cosmid clones from chromosome 11. Several rare cutting restriction enzyme sites were found in close vicinity to the translocation breakpoint, and a long-range map spanning 1000 kb of chromosome region 11p13 was established. Analysis of the DNA from 15 cases of sporadic and familial Wilms' tumor did not reveal any changes, indicating that the translocation breakpoint does not reside in this gene.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

α-Amylase structural genes in rye

Summary Rye -Amy1, -Amy2, and -Amy3 genes were studied in the cross between inbred lines using wheat -amylase cDNA probes. The -Amy1 and -Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the -Amy3 region was much more conserved. The numbers of restriction fragments found and the F₂ segregation data suggest that there are three -Amy1 genes, two or three -Amy2 genes, and three -Amy3 genes in rye. These conclusions were supported by a simultaneous study of -amylase isozyme polymorphism. The F₂ data showed the three individual -Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two -Amy2 genes were shown to be separated by 5 cM. Linkage data within -Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

α-Globin gene deletion causes α-thalassemia syndromes in two German families

Summary Restriction endonuclease mapping of chromosomal DNA has been used to determine whether the -globin gene deletion or non-deletion form of -thalassemia is the underlying molecular defect in individuals of two unrelated German families with -thalassemia syndromes. The obtained DNA pattern in all cases indicated loss of -globin genes resulting in-/,--/, and--/- genotypes in thalassemia-2, -thalassemia-1, and Hb H individuals respectively. The chromosomes showing loss of one -globin gene in -thalassemia-2 and Hb H disease were characterized by the so-called rightward deletion form exhibiting loss of a 3.7 kb DNA fragment in the -gene cluster.     

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.     

Characterization of T cell clones specific to a determinant of hepatitis B virus core and e antigens in chronic type B hepatitis: Implication for a T cell mechanism of HBV immunopathogenesis

T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130–140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) chain variable (V) gene was found: V12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR chain joining region (J) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V gene usage and the expression of conserved residues in junctional-region sequences of TCR chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis.     

Expression of enzymatically active,recombinant barley α-glucosidase in yeast and immunological detection of α-glucosidase from seed tissue

An -glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae -factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant -glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid -glucosidase. The enzyme had a K_m of 1.88 mM and V_max of 0.054 µmol/min on maltose. The recombinant -glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley -glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa -glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in -glucosidase enzyme activity.Abbreviations: AGL, barley seed -glucosidase; rAGL, recombinant barley seed -glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.