Loss of the Y chromosome from normal and neoplastic bone marrows. United Kingdom Cancer Cytogenetics Group (UKCCG)

Loss of the Y chromosome is a feature of haematologically normal bone marrow in elderly males, but it is also found in haematological malignancy. We describe -Y as the sole karyotypic abnormality in 147 cases (66 from 802 unselected cases and a further 81 cases selected for -Y) with the following diagnoses: no haematological malignancy (N), myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and myeloproliferative disorder (MPD). The frequency of -Y in the 802 unselected N, MDS, and AML cases was 7.7%, 10.7%, and 3.7%, respectively. It could not be evaluated in MPD because there were too few cases. In N and MDS cases the frequency increased in a similar fashion over the age of 60 years. The 147 -Y cases showed a similar increase in distribution with advancing age in all four clinical categories. The degree of loss of Y (% -Y cells per patient) increased with age in N and MPD patients but not in those with MDS or AML. This study suggests that in elderly men -Y is not indicative of malignancy and should not be considered as a marker of the malignant clone.


Detection of loss of heterozygosity at the human TP53 locus using a dinucleotide repeat polymorphism.

Loss of heterozygosity at the TP53 locus occurs frequently in many types of cancer and requires polymorphic markers for detection. Several polymorphisms at the TP53 locus have been described previously, and polymerase chain reaction (PCR)-based assays have been developed to detect these polymorphisms. However, these polymorphisms have relatively low levels of heterozygosity and are often uninformative. We report here the detection of loss of heterozygosity at the TP53 locus in various human cancers by using a highly informative dinucleotide repeat polymorphism.


Deletions in the short arm of chromosome 8 are present in up to 90% of human colorectal cancer cell lines.

Cytogenetic analyses of human colon cancer cells have revealed non-random deletions in chromosome arm 8p, among other chromosomal changes. By using 8p-specific DNA probes we could identify allelic loss in 87% of colon cancer cell lines. Corresponding analyses in direct preparations of colon tumor tissues revealed a minimal value of 40% of allelic loss but were obstructed in many instances by contaminating normal tissue. These findings add to the number of non-random genetic alterations occurring during colon carcinogenesis.


Is cancer cytogenetics reducible to the molecular genetics of cancer cells?

Whether cancer cytogenetics can be reduced to the molecular genetics of cancer cells is a question that must be addressed in three domains, focusing on its ontological, methodological, and epistemological dimensions. The possibility of ontological reduction hinges on whether chromosomes have other important constituents than molecules. Although this must obviously be answered in the negative, it should be emphasized that both cytogenetic and recombinant DNA investigations provide us with very selective pictures of genomic organization. This is of concern because the higher order packing of DNA and its joining with other molecules to form chromosomal structures give rise to emergent properties, functional features that become manifest only at higher levels of complexity and that may not be deducible from the base pair composition of the DNA. A position of extreme methodological reductionism would in our context be that the best research strategy is always to investigate the genetic changes of tumor cells at the highest possible resolution level, as alterations of genes and, ultimately, as changes in DNA primary structure. There are two fundamental differences between cytogenetic and molecular genetic techniques that make this stance untenable. First, whereas cytogenetic investigations are open-framed (all chromosome aberrations are revealed), molecular genetic analyses are highly specific (only those aberrations are revealed that one tests for). Second, whereas the molecular approach determines the genotypic constitution of an idealized, average tumor cell, cytogenetic analysis is of real, individual cells. These may not necessarily be representative of the main population of the tumor, but at least whatever karyotypic differences exist between them are detected. Heterogeneity and clonal evolution within the tumor can thereby be assessed.(ABSTRACT TRUNCATED AT 250 WORDS)


Non-random abnormalities of chromosomes 3, 6, and 8 associated with posterior uveal melanoma.

We present ten cases of posterior uveal melanoma which were karyotyped after short-term culture. One tumour had a normal chromosome complement. The remaining nine tumours were cytogenetically abnormal, with chromosomes 3, 6, 8, 11, and 13 most frequently involved. Abnormalities of chromosome 13 were seen in two cases, chromosome 11 in three cases, and chromosomes 3, 6, and 8 in five cases. Four tumours, all derived from the ciliary body, demonstrated monosomy 3 and i(8q), confirming the involvement of these aberrations with a subgroup of uveal melanomas arising from the ciliary body.


Molecular characterization of chromosome 22 deletions in schwannomas.

Schwannomas are tumors of the cranial, spinal, and peripheral nerve sheaths that originate from Schwann cells. Acoustic neurinomas are the most frequent cranial schwannomas. They might develop sporadically or in the context of neurofibromatosis type 2 (NF2). Loss of part or all of chromosome 22 is frequently found in acoustic schwannomas, suggesting that the NF2 gene is a tumor suppressor gene involved in the genesis of these tumors. Only a few spinal schwannomas have been molecularly characterized so far, showing that chromosome 22 loss might also occur in these tumors. Here we present the molecular analysis of chromosome 22 in 23 acoustic schwannomas and nine schwannomas of other locations (including other cranial nerves and spinal and peripheral nerves). Most of these tumors were from sporadic cases. Multiple schwannomas of various locations were analyzed in two patients with NF2. We found partial or complete monosomy for chromosome 22 in 22% of the acoustic schwannomas and 55% of the non-acoustic schwannomas. The tumors with partial monosomy included four with terminal deletions and one with a deletion of the centromeric part of the long arm of chromosome 22. The region between the beta B2-1 crystallin locus (CRYB2A) and the myoglobin locus (MB) was commonly deleted in these tumors. Our studies suggest that a schwannoma-related tumor suppressor gene within this region, which might be the NF2 gene, is involved in the development of schwannomas of various locations in the nervous system. Our studies indicate that the second hit in the genesis of different schwannomas within one (predisposed) NF2 patient occurs independently and via different mechanisms.


Cytogenetic studies on 19 papillary thyroid carcinomas.

Short-term cultures from 19 papillary thyroid adenocarcinomas revealed clonal numerical and/or structural chromosomal changes in 13 tumors, nonclonal abnormalities in one tumor, and only normal karyotypes in five tumors. Clonal abnormalities of chromosome 10 were present in three tumors, two of which had the translocation t(7;10)(q35;q21). Numerical abnormalities of chromosome 17 were detected in two tumors.


Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas.

A combined cytogenetic and molecular analysis was performed on 11 cases of papillary thyroid carcinoma. A simple karyotypic abnormality was detected in five tumors, whereas six had no apparent chromosome change. In four of five rearranged cases the presence of a specific chromosomal abnormality involving chromosome 10 (cases 1 and 2) and chromosome 1 (cases 3 and 4) was associated with the rearrangement of two protooncogenes: RET and NTRKI (formerly trk), respectively, with different donor genes. Moreover, the chromosomal localization of the involved genes and the type of chromosomal change observed suggested that RET and NTRKI activation occurred by intrachromosomal rearrangements. The six cases with normal karyotype did not show RET or NTRKI activation. These findings suggest that a combined cytogenetic and molecular approach would be useful in understanding the pathogenesis of thyroid neoplasia.


Preferential chromosome loss in human papilloma virus DNA-immortalized mammary epithelial cells.

Human papilloma virus (HPV) DNA-immortalized human mammary epithelial cells may provide a model system for studying the molecular basis of immortalization and its role in breast neoplasia. Cytogenetic analyses were performed on clones derived from HPV 16- and HPV 18-immortalized human mammary epithelial cells. The majority of the clones contained near-diploid karyotypes. The single most frequent whole-chromosome loss was that of chromosome 19. Regions that showed preference for deletion and/or translocation included 2pter, 11qter, and 15pter. Evidence of chromosome 19 loss was confirmed by polymerase chain reaction (PCR)-generated, chromosome 19-specific, dinucleotide microsatellite repeat polymorphism analysis.


Characterization of the translocation breakpoint sequences of two DEK-CAN fusion genes present in t(6;9) acute myeloid leukemia and a SET-CAN fusion gene found in a case of acute undifferentiated leukemia.

The t(6;9) associated with a subtype of acute myeloid leukemia (AML) was shown to generate a fusion between the 3' part of the CAN gene on chromosome 9 and the 5' part of the DEK gene on chromosome 6. The same part of the CAN gene appeared to be involved in a case of acute undifferentiated leukemia (AUL) as well, where it was fused to the SET gene. Genomic sequences around the translocation breakpoint were determined in two t(6;9) samples and in the case of the SET-CAN fusion. Although coexpression of myeloid markers and terminal deoxynucleotidyl transferase was shown to be one of the characteristics of t(6;9) AML, no addition of random nucleotides at the translocation breakpoint could be found. In addition, the breakpoint regions did not reveal heptamer-nonamer sequences, purine-pyrimidine tracts, a chi-octamer motif, or Alu repeats. The sequence in which the translocation breakpoints occurred was enriched in A/T. Notably, the specific introns in which clustering of breakpoints occurs in DEK and CAN both contain a LINE-I element. As LINE-I elements occur with a moderate frequency in the human genome, the presence of such an element in both breakpoint regions may be more than coincidental and may play a role in the translocation process.


Whole-arm t(1;16) and i(1q) as sole anomalies identify gain of 1q as a primary chromosomal abnormality in breast cancer.

Cytogenetic analysis of four ductal breast carcinomas revealed net gain of 1q in all tumors. In the first tumor, the only change was that one chromosome 16 was replaced by a derivative chromosome consisting of 16p and 1q. The same unbalanced whole-arm translocation was also found in the second tumor, as the only aberration in one of four abnormal clones. In the last two cases, which also were characterized by cytogenetically unrelated clones, an extra i(1q) was present in one clone in both tumors as the sole aberration. Our findings suggest that gain of 1q is a primary chromosomal abnormality in breast carcinomas, in the sense that it is an early event that precedes the acquisition of more complex changes.


Recurrent abnormalities of chromosome bands 10q23-25 in non-Hodgkin's lymphoma.

Many nonrandom chromosome abnormalities have been associated with non-Hodgkin's lymphomas (NHL). Some of these are nonspecific changes seen in many different histologic subtypes. We describe a series of abnormalities of chromosome bands 10q23-25 seen in 159 consecutive NHL patients with abnormal cytogenetic findings. The proportion of karyotypes with abnormalities of 10q varied from 3% among the immunoblastic lymphomas to 67% in the diffuse large cleaved cell lymphomas. Seventeen (10.7%) had abnormalities of 10q23-25. All but one of these were B-cell tumors. The abnormalities consisted of six deletions and 11 translocations. Sixteen of the 17 patients had the 10q abnormality when cells were first karyotyped. The remaining patient acquired the 10q abnormality in the third of a series of biopsies. In the follicular histologic subtypes [follicular small cleaved cell (FSC), follicular mixed small cleaved and large cell (FM), and follicular large cell noncleaved (FL-NC)], abnormalities of 10q were found in nine patients, all in association with abnormalities of 14q32. Seven of these were associated with the t(14;18)(q32;q21). Overall, 10q23-25 abnormalities were observed in 11.9% (8/67) of low-grade [small lymphocytic (SL), FSC, and FM] lymphoma cases. DNA was available from five patients with abnormalities of 10q and was probed for rearrangements with the HOXII (TCL3) oncogene probe. As expected, we did not find such rearrangements in these five patients with B-cell tumors. Abnormalities of 10q23-25 have been reported previously in NHL but not at this frequency.


Molecular cloning and analysis of chromosome band 11q23 involved in leukaemia-associated translocations.

Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised. The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11q23, and a restriction enzyme map of this region has been constructed. The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined. It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11). The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy. It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.


Translocations involving 12p in acute myeloid leukemia: association with prior myelodysplasia and exposure to mutagenic agents. United Kingdom Cancer Cytogenetics Group (UKCCG).

Six cases of acute myeloid leukemia (AML) with translocations involving 12p are described. The patients were one child age 7 yrs and five adults with an age range of 20-66 yrs (median 46 yrs). In two patients AML followed treatment for acute lymphoblastic leukemia (ALL), in one case after 11 years disease-free survival. Of the remaining four patients, two had been occupationally exposed to possible mutagens and three had a previous myelodysplastic phase. Two patients achieved complete remission; survival for the six cases was between 1 and 24 months (median 6.5 months). The breakpoints in 12p occurred in p11, p12, and p13, indicating that several sites are important in these rearrangements, and it is suggested that t(12;17)(p11;q11) is a new nonrandom abnormality in AML.


Translocations involving 9p and/or 12p in acute lymphoblastic leukemia. United Kingdom Cancer Cytogenetics Group (UKCCG).

Fifteen cases of acute lymphoblastic leukemia (ALL) with translocations involving 9p and/or 12p are described. Four children, three males and one female, age range 1-8 yrs, had translocations involving 9p but not 12p. Of these, three had B-lineage ALL and one had biphenotypic T-ALL/acute myeloid leukemia. Six patients, three males and three females, age range 1-49 yrs, had translocations involving 12p but not 9p. Five had B-lineage ALL and one had T-ALL. One patient had dic(7;12)(p11;p11), confirming that this previously reported translocation is nonrandom in ALL. One patient had t(2;12)(q14;p13), and it is suggested that this may also be a new nonrandom abnormality. Five patients, four males and one female, age range 11-21 yrs, had common ALL and dic(9;12). The dic(9;12) appears to confer a better prognosis than do other translocations involving 9p or 12p.


Clonal 6p21 rearrangement is restricted to the mesenchymal component of an endometrial polyp.

Previous cytogenetic analyses revealed t(6;20)(p21;q13) in two endometrial polyps. We karyotyped a large endometrial polyp in which 19 of 25 metaphase cells contained a t(1;6;4)(q21;p21;q13). Subsequent combined immunohistochemical/cytogenetic analysis showed all aberrant metaphase cells to be of mesenchymal derivation, whereas epithelial cells from the polyp were diploid. These studies indicate that rearrangement of chromosome band 6p21 is a characteristic cytogenetic aberration in the stromal component of endometrial polyps.


The der(11) chromosome contains the critical breakpoint junction in the 4;11, 9;11, and 11;19 translocations in acute leukemia.

Translocations involving 11q23 are recurring abnormalities in human acute leukemia cells of either lymphoid or myeloid lineage. Analysis of 13 variant translocations associated with four of these [t(4;11), t(6;11), t(9;11) and t(11;19)] reveals that the der(11) chromosome is conserved in all of them and therefore contains the critical genetic rearrangement. The MLL gene (myeloid/lymphoid leukemia) is involved in each of these translocations. It is transcribed from centromere to telomere. The present analysis indicates that the 5' region of MLL on the der(11) is juxtaposed to the coding sequences of genes on each of the other translocation partners.


Variant translocations in two Burkitt's lymphoma cell lines are located in the MLV14 locus.

The human MLV14 locus, located 20 kilobases 3' of MYC, is rearranged in two Burkitt's lymphoma cell lines with either a t(2;8)(p12;q24) or t(8;22)(q24;q11). Alterations of MLV14 may have prognostic significance in some types of B-cell malignancies.


Expression of the CD2 molecule on human B lymphoid progenitors.

CD2 expression on human B lymphoid progenitor cells was examined. By immunofluorescence analysis, a small fraction of bone marrow B cells was found to express CD2 on their surface. CD2 expression was not demonstrated on peripheral B cells. Epstein-Barr virus-transformed B cell lines derived from fetal liver at 8 weeks of gestation were analyzed to delineate the expression and function of CD2 at the early stage of human B cell development. Characterization of surface and genomic phenotypes of cell lines revealed that the established cell lines represent at least three different phenotypic characteristics of early B lineage cell: B progenitor, pre B, or early B cell. None of the 18 cell lines and 13 subclones with the phenotype of the early B lymphoid cells initially expressed CD2 antigen. However, CD2 expression was induced by the successive cultivation of some cloned B progenitor cell lines. In spite of the expression of CD2, these clones cell lines were unable to form rosettes with sheep red blood cells. By immunoprecipitation analysis, an identical 50 kDa protein was precipitated with anti-CD2 antibody from the lysates of the radioiodinated CD2+B progenitor cell line and peripheral blood T cells. Anti-CD2 antibody induced significant enhancement of proliferation of the CD2+B progenitor subline. These data indicate that human CD2 is expressed on a fraction of B lineage cells at a very early differentiation stage and may play a role in B lymphopoiesis.


Peptide-conjugated hapten groups are the major antigenic determinants for trinitrophenyl-specific cytotoxic T cells.

Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC molecules ('hapten-peptide determinants'). However, cell modification with trinitrobenzene sulfonic acid (TNBS) also leads to the formation of TNP determinants covalently attached to MHC molecules ('altered self'). To determine the importance of 'peptide' versus 'altered self' determinants, we used the mutant cell line RMA-S which expresses peptide-free ('empty') Kb and Db molecules at 26 degrees C. Additionally, we stabilized Kb molecules on RMA-S cells at 37 degrees C using the Kb binding heptapeptide N53-59 derived from the vesicular stomatitis virus nucleoprotein. Lacking lysine, this peptide remains unmodified by TNBS and, therefore, only allows the formation of 'altered self' TNP determinants on occupied Kb molecules. RMA-S targets, pretreated or untreated with N53-59, upon TNBS modification were only lysed poorly or not at all by four different TNP-specific CTL. In contrast, all of these clones efficiently lysed TNBS-treated, unmutated RMA cells, and three of them strongly reacted with RMA or RMA-S cells in the presence of tryptic TNP-BSA peptides. Moreover, the clone unreactive for TNP-BSA peptides also recognized TNP self-peptides extracted from TNBS-treated syngeneic spleen cells. Taken together, these data clearly show that TNP residues linked to MHC via associated peptides but not by covalent bondage represent the dominant antigenic epitopes for class I MHC-restricted, hapten-specific T cells.


Differential expression of three CD45 alternative structures on murine T cells: exon 6-dependent epitope as a marker for functional heterogeneity of CD4+ T cells.

We prepared a novel rat mAb specific for CD45 molecules bearing the epitope coded for by the alternative exon 6 of the murine CD45 gene (CD45RC). Together with available mAbs to alternative exon 4- and 5-dependent epitopes (CD45RA and CD45RB respectively), we found that the three alternative exons show differential expressions on murine lymphocytes. Flow cytometry analysis revealed that although B cells were homogeneously CD45RA+B+C+, the CD4+ T cells clearly included two populations, CD45RA-B+C- and CD45RA-B+C+. The CD8+ T cells were separated into CD45RA-B+C+ and CD45RA+B+C+ populations. Such features of epitope expression on the cell surface correlate well with message levels of corresponding alternative exons. In the CD4+ T cells, messages of alternative exons were associated with either one or two exon forms of the CD45 transcript. In CD8+ T cells, there were transcripts with one, two, or three alternative exons. When stimulated by an immobilized CD3 mAb, the CD45RC+CD4+ T cell subset preferentially secreted IL-2 and CD45RC-CD4+ T cells produced IL-4. Upon stimulation with concanavalin A, CD45RC-CD4+ T cells converted to CD45RC+ cells, and the level of CD45RC expression on the CD45RC+CD4+ T cell subset was up-regulated. These changes were unidirectional and irreversible. Therefore, differential expression of CD45RC probably delineates the functional heterogeneity of murine CD4+ T cells that is associated with the stages of CD4+ T cell maturation or activation.


Evidence against a pathogenetic role for endothelin in pre-eclampsia.

To assess whether increased placental or systemic endothelin synthesis has a pathogenic role in pre-eclampsia (gestational proteinuric hypertension). Prospective observations study. 19 women with pre-eclampsia and 10 healthy pregnant women were studies. All were in the last trimester. Preproendothelin-1 gene expression by Northern blot analysis and generation of endothelin-1 precursor, big-endothelin-1, and endothelin isoforms, namely endothelin-1, 2 and 3, were assessed by specific radio-immunoassays, in placental tissue. Plasma endothelin-1 levels and urinary excretion of big-endothelin-1 and endothelin-1 were measured. Placental preproendothelin-1 gene expression and immunoreactive big-endothelin-1 and endothelin-1, 2 and 3, were comparable in placental tissue from pre-eclamptic and normal pregnant women. Plasma levels of endothelin-1 did not differ between pre-eclamptic and normal pregnancies. In contrast, urinary excretion of endothelin-1, which is likely to reflect the renal synthesis of the peptide, was significantly decreased in pre-eclamptic, as compared with normal pregnant women. This was not due to a decreased renal generation of endothelin-1 precursor, since urinary excretion of big-endothelin-1 did not differ between pre-eclamptic and normal pregnancies. These data suggest an increased renal endothelin-1 breakdown in pre-eclampsia. Endothelin is unlikely to play a role in the pathogenesis of pre-eclampsia. Instead, an increased renal breakdown may have a role in limiting the negative effects of other vasoactive factors on the renal circulation.


[Study on the therapeutic effects of interferon and gamma-globulin in experimental Pneumocystis carinii pneumonia].

This study was performed to observe the therapeutic effects of interferon-gamma(IFN-gamma) and gamma-globulin(gamma-globulin) in experimental Pneumocystis carinii pneumonia of immune suppressed mice. After 9 weeks, trimethoprim-sulfamethoxazole(TMP-SMZ; 10-50 mg/mouse/day), mouse IFN-gamma(5 x 10(4) units/mouse/day) and mouse gamma-globulin(20 mg/mouse/day) were administered to the mice for 3 weeks by the experimental group. The therapeutic efficacy was evaluated by body weights, histopathologic and electron microscopic findings of the lungs, and number of P. carinii cysts by Gomori's methenamine silver stain. Body weights of the mice were significantly increased in the group of combination therapy of TMP-SMZ with IFN-gamma or gamma-globulin, and in the group of TMP-SMZ treatment (p < 0.05), however, little effect was found in the group of gamma-globulin alone. Histopathologic findings of P. carinii pneumonia were much improved in the group of combination therapy of TMP-SMZ with IFN-gamma. Treatment with either TMP-SMZ or IFN-gamma significantly reduced the number of cysts in the P. carinii pneumonia, but gamma-globulin alone was ineffective. In electron microscopic findings of P. carinii pneumonia, the number of trophozoites and cysts were reduced by treatment with either TMP-SMZ or IFN-gamma, and most of the cysts were empty or containing one or two intracystic bodies. The present results suggested, that combination therapy of TMP-SMZ with IFN-gamma had synergistic effects in treatment of P. carinii pneumonia in experimental mice.


Bilateral congenital mydriasis.

A single case of bilateral congenital mydriasis is described. A review of the literature is presented and possible modes of inheritance are discussed.


Idiopathic choriovitreal membrane--a case report.

A case of a macular idiopathic choriovitreal membrane is described which developed in a diabetic man. On initial examination the patient was found to have a pigment epithelial detachment with a choroidal neovascular membrane (CNVM) in the right eye. Two months after the first visit the CNVM was seen to have penetrated the retina and presented as a choriovitreal membrane. Panretinal photocoagulation was applied after which the choriovitreal membrane demonstrated fibrotic involution. This case is unusual in that the choriovitreal membrane developed in the absence of a choroidal or retinal pigment epithelial disease process that may be associated with a CNVM as well as in the absence of previous macular laser treatment.


Embryonal form of extraskeletal myxoid chondrosarcoma with intermediate filament positive hyaline-like globules.

Reported is a case of an embryonal form of extraskeletal myxoid chondrosarcoma. The tumor cells contained PAS-positive, eccentrically located intracytoplasmatic, hyaline-like globules. These globules seemed to be a hallmark of this tumor, and they reacted strongly with cytokeratin and S-100 protein antibodies. The tumor was negative in reactions with desmin and GFAP antibody.


S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.


RNA binding assays for Tat-derived peptides: implications for specificity.

RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.


Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.

Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.


Effects of pH on bacterial porin function.

Porin is a trimeric channel-forming protein in the outer membrane of Gram-negative bacteria. Functions of the porins OmpF, OmpC, and PhoE from Escherichia coli K12 were analyzed at various pHs. Preliminary results from bilayer lipid membrane and liposome swelling assays indicated that in vitro porin has at least two open-channel configurations with a small and a large size. The small channels were stabilized at low pH while the larger channels were detected under basic conditions. The size switch occurred over a very narrow range near neutral pH, and the two major open-channel configurations responded differently to variations in voltage. The presence of two or more pH-dependent substates of porin could explain the variability in pore diameter measured by others and suggests a more dynamic role for porin in the cell.


Involvement of histidine-21 in the pH-induced switch in porin channel size.

Porin is a channel-forming protein in the outer membrane of Gram-negative bacteria. In the previous paper (Todt et al., 1992), we showed that the pH induced a switch in the channel size in vitro for the porins OmpF, OmpC, and PhoE. In the results presented here, His21 of OmpC and OmpF from Escherichia coli was chemically modified with diethyl pyrocarbonate. Functional analysis of these modified porins at different pHs suggested that this histidine is involved in the pH-induced switch in channel size. Secondary structure analysis of porins at various pHs using Fourier transform infrared spectroscopy indicated that there was no global change in structure accompanying the pH-induced switch in channel size.


Effect of antibody binding on protein motions studied by hydrogen-exchange labeling and two-dimensional NMR.

We have used hydrogen-exchange labeling detected by 2D NMR to study antibody-protein interactions for two monoclonal antibodies raised against horse cytochrome c. The data show that these antibodies bind mainly to the large 37-59 omega-loop of the cytochrome c molecule. In addition, the results provide some suggestive evidence concerning units of local structural flexibility in cytochrome c.


Stereoelectronic factors in the interaction with DNA of small aromatic molecules substituted with a short cationic chain: importance of the polarity of the aromatic system of the molecule.

We have performed a quantitative analysis of the interaction with DNA of several unfused aromatic compounds synthesized in our laboratory and substituted with one or two short cationic chains. These and similar literature compounds, for which DNA binding data are available, bind with DNA by partial intercalation of the aromatic system, groove interaction of the linker chain, and groove electrostatic interactions of the terminal cationic group. Several independent quantitative and qualitative approaches show consistently that the strength of the interaction of the aromatic unit of the molecule with DNA binding sites depends on the direction and magnitude of polarity of the aromatic system. The phenomenon is explained in terms of the greatest negative potential in the DNA grooves, a concept extensively elaborated by Pullman and Pullman [cf. Lavery, R. and Pullman, B. [(1985) J. Biomol. Struct. Dyn. 2, 1021-1032] and references therein]. Classical, fused-ring planar intercalators do not follow the polarity-DNA affinity correlation, presumably because the intercalative forces depend more strongly on polarizability than on polarity of the aromatic system.


Endonucleolytic cleavage of a long 3'-trailer sequence in a nuclear yeast suppressor tRNA.

Transcripts of Saccharomyces cerevisiae nuclear tRNA genes are normally terminated within a few nucleotides of the tRNA coding region, in contrast to mitochondrially encoded tRNAs, which are contained within polycistronic transcripts and thus require 3'-processing by mitochondrial endonucleases. We show that 3'-processing activities capable of removing artificially extended 3'-trailer sequences from some tRNA substrates are also present in the yeast nucleus. Correct 3'-processing in vivo resulted in the formation of functional suppressor tRNA. The 3'-processing activities were also identified in vitro through analysis of transcription-processing products in cell-free yeast S-100 extracts. Comparison of several pre-tRNA substrates showed that the tRNA structure played a major role in determining the processability of a substrate but that the nature of the 3'-trailer sequence also modulated the rate of 3'-processing. Pre-tRNA containing mitochondrial tRNA(Val) sequence was a good substrate for in vitro processing, independent of its 3'-trailer. A 200-nt-long pre-tRNA, encoding the nuclear SUP4 tRNA gene and a mitochondrial 3'-trailer, was processed in yeast S-100 extract in a multistep pathway into mature-sized tRNA(Tyr). Part of the 3'-processing was due to an endonuclease which cleaved near or precisely at the 3'-end of the coding region of the tRNA. A short sequence around this endonucleolytic 3'-cleavage site was crucial for the formation of active suppressor tRNA in vivo. A 9-nt-long sequence motif derived from the mitochondrial 3'-trailer allowed processing, while sequences derived from lacZ or pBR322 DNA were processed neither in vitro nor in vivo.


Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA.

Four previously undetermined sites of methylation are mapped in Escherichia coli 23S rRNA employing a novel combination of methods. First, using a double-isotope approach, the total number of methyl groups in 23S rRNA was determined to be 14.9 +/- 1.6. Second, hybridization of methyl-labeled rRNA to complementary DNA restriction fragments and PAGE analysis were used to purify RNA-DNA heteroduplexes and to quantify methyl groups within specific 23S rRNA fragments. Third, the methylated nucleosides in these fragments were identified and quantified using HPLC, confirming the presence of 14 methylation sites in 23S rRNA, four more than had been previously identified. In contrast, a similar set of analyses conducted on 16S rRNA gave evidence for 10 sites of methylation, at all approximate locations consistent with published 16S methylated nucleoside identities and locations. Selected regions of the 23S rRNA molecule containing previously unidentified methylated nucleosides were released by site-directed cleavage with ribonuclease H and isolated by PAGE. Sites of methylation within the RNA fragments were determined by classical oligonucleotide analyses. The four newly identified methylation sites in 23S rRNA are m2G-1835, m5C-1962, m6A-2503, and m2G at one of positions 2445-2447. Together with previously described sites of modification, these new sites form a group that is clustered in a current model for the three-dimensional organization of the 23S rRNA in the 50S ribosomal subunit, at a locus congruent with nucleotides previously implicated in ribosomal function.


Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.


Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.


Induction of two K+ currents by complement component C5a in mouse macrophages.

Puff application of complement component C5a (5 x 10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced two kinds of outward current at a holding potential of -68 mV, a slowly-rising sustained outward current and a spike-like transient outward current. Quinidine (2 x 10(-4) M) and tetraethylammonium (10(-2) M) partially suppressed both types of outward current. Charybdotoxin (2 x 10(-6) M) markedly suppressed the spike-like outward current. Reversal potentials in bath solutions of different external K+ concentrations were dependent only on K+ concentrations. The transient current was not suppressed in Ca(2+)-free EGTA-containing solution, but was completely abolished in BAPTA-containing solution. One kind of single channel responding to C5a, which has a single-channel conductance of 29 pS, was recorded from cell-attached patches. These results suggest that C5a activates a Ca(2+)-dependent and another type of K+ current.


Interaction of melittin derivatives with lipid bilayer membrane. Role of basic residues at the C-terminal and their replacement with lactose.

Melittin possesses an amphiphilic property in the primary sequence in which hydrophilic residues are located at the C-terminal region from Lys-21 to Gln-26. A part of the hydrophilic sequence was cleaved off by endopeptidase Arg-C to obtain melittin 1-22. The affinity of melittin 1-22 for neutral phospholipid membrane was reduced to 1/3 that of melittin, indicating that the basic residues, Lys-23 and Arg-24, are important in binding of melittin to the membrane. The melittin 1-22 was extended toward the C-terminal end by connection of lactose (melittin-lac), the membrane affinity of which was slightly higher than the melittin 1-22, but lower than melittin. The leakage experiment of 5,6-carboxyfluorescein encapsulated in DPPC liposomes showed that the activities of melittin 1-22 and melittin-lac in membrane lysis were much lower than melittin. However, the melittin 1-22 formed a voltage-dependent ion-channel in an azolectin bilayer membrane. It is thus considered that Lys-23 and Arg-24 residues of melittin play an important role in binding to the polar region of membrane for lysis, but not for ion-channel formation.


Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.


A patch-clamp study of Bacillus subtilis.

In patch-clamp experiments on giant protoplasts of the Gram-positive bacterium Bacillus subtilis, membrane stretch resulted in an initial transient collapse of the membrane resistance, after which stretch-activated, voltage modulated, high-conductance channels could be observed. The channel open probability increased exponentially with applied suction and positive voltage, as a result of variations of both the mean open and the mean closed times. The substate structure and other characteristics of the electrical activity suggested the presence of a family of pores exhibiting cooperative behavior. A role in osmotic protection is suggested. In the intact bacteria, the pores may be part of an unidentified envelope apparatus, having other functions as well.


Expression of cell adhesion molecules in human cardiac allograft rejection.

Adhesion of leukocytes to vascular endothelial cells is a critical step in a variety of inflammatory conditions. We studied the expression and distribution of intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) in frozen sections of 83 endomyocardial biopsy specimens from human allograft hearts using monoclonal antibodies and an avidin-biotin complex-alkaline phosphatase staining technique. Cases with cellular or humoral rejection and Quilty lesions were studied. Staining was graded from 0 to 3+ in lymphocytes and in capillary, arterial, venular, and endocardial endothelial cells. Expression of ICAM-1 in capillaries increased with the severity of cellular rejection and was prominent in humoral rejection. ICAM-1 was also expressed in lymphocytes in proportion to the degree of rejection. Little or no ELAM-1 expression was noted. In Quilty lesions the intensity of ICAM-1 expression was similar to that of mild-to-moderate rejection. Thus adhesion molecule expression can be identified in endomyocardial biopsy specimens of patients with rejection, suggesting a role for adhesion molecules in the process of rejection. These findings may prove useful in monitoring rejection and its response to therapy and in developing specific antisera directed against these molecules.


Development of a procedure for coupling the homing device glu-plasminogen to liposomes.

The aim of this study was to find a suitable way of coupling the homing-device glu-plasminogen to the outside of liposomes. The described procedure is based on the reaction of thiol-groups introduced in the protein with thiol-reactive groups of the liposome. Details on the thiolation of proteins with the reagent succinimidyl-S-acetylthioacetate (SATA) were studied for a model-protein, amylase. Increasing the incubation-ratio SATA: amylase resulted in a gradually growing number of introduced thiol-groups, until a maximum of about 5 mol SH per mol amylase was reached. The enzymatic activity of the derivatized protein was even higher than that of native amylase. The thiol-introduction was then applied to glu-plasminogen itself. After activation with SATA, the protein was incubated with liposomes containing the thiol-reactive anchor maleimido-4-(p-phenylbutyrate)-phosphatidylethanolamine (MPB-PE). Under the chosen conditions, incubation of 0.5-2.5 mg/ml protein with 6.0-7.5 mumol/ml phospholipid for 30-120 min resulted in coupling-ratios of 20 to 94 micrograms glu-plasminogen per mumol phospholipid. This corresponds with about 140 to 660 protein molecules per liposome. SATA-derivatization of glu-plasminogen brought about a loss of its enzymatic activity induced by streptokinase. This activity of liposomally coupled plasminogen was about 52 to 74% of the activity of native glu-plasminogen (depending on the coupling-ratio). Although this may seem a significant loss of activity, it was shown that the capacity of liposomal glu-plasminogen to bind to its target, fibrin, was not reduced but several fold higher under the used conditions than that of the free protein. Therefore, the described method for thiol-introduction is an effective way to thiolate amylase without loss of activity, and to bind the homing-device glu-plasminogen to liposomes without substantially interfering with its fibrin-binding/homing capacity.


Binding of a monoclonal antibody E12 to Gc globulin (vitamin D-binding protein) is inhibited by actin.

A monoclonal antibody, E12, to human Gc globulin was raised in murine somatic cell using purified Gc. The antibody was subtyped IgG2b kappa and had a kd of 3.0 x 10(-8) M for antigen Gc. Monospecificity for Gc was demonstrated by Western blotting of normal human serum using nondenaturing polyacrylamide gel electrophoresis. As judged by ELISA, actin inhibited binding of E12 to Gc in dose-dependent fashion. Affinity chromatography studies further showed that ternary complexes of actin-Gc-E12 were not formed, and actin displaced Gc from Gc-E12 complexes. Proteolytic digestion of Gc with trypsin showed that the monoclonal antibody E12 reacted with the major 30-kDa tryptic fragment containing the amino terminal fragment of Gc, but actin did not react with this fragment. These results indicate that interaction of actin with Gc causes conformational changes which inhibit binding of E12.


Transfer RNA genes from the hyperthermophilic Archaeon, Methanopyrus kandleri.

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA(Gln) and tRNA(Lys) genes. All four of the tRNA genes appear to encode the 3' terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98 degrees C optimum growth temperature of M. kandleri.


Excessive production of transforming growth-factor beta 1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth.

Angiogenesis is an important part of tumor growth in vivo. We used the transfected Chinese hamster ovary (CHO) cells that overproduced recombinant transforming growth-factor beta 1 (TGF-beta 1) to examine the possible role of this factor in tumor growth and angiogenesis in a nude mouse model. The in-vitro proliferation of TGF-beta 1-transfected CHO cells was unaffected by the treatment of either recombinant TGF-beta 1 or an anti-TGF-beta 1 antibody. The TGF-beta 1-transfected cells grew more rapidly than the parental CHO cells when injected subcutaneously into nude mice. The tumors derived from the TGF-beta 1-transfected cells showed prominent tumor-associated angiogenesis, whereas the parental cells produced tumors without such angiogenesis. In addition, an anti-TGF-beta 1 neutralizing antibody was able to inhibit both growth and angiogenesis in the tumors derived from TGF-beta 1-transfected cells. These findings suggest that the overproduction of TGF-beta 1 by tumor cells can contribute to neovascularization and may help promote tumor development in vivo.


Purification of an active receptor for acidic and basic fibroblast growth factor from bovine retina.

Acidic and basic fibroblast growth factors (FGFs) influence cell division and differentiation in retina cells. Their effects are thought to be mainly mediated through stimulation of a specific membrane receptor and subsequent generation of an intracellular signal pathway. In this study, we purified a FGF receptor of 130 kDa from bovine neural retina using wheat germ agglutinin affinity chromatography followed by FGF-affinity chromatography. The isolated receptor showed ligand binding activity with dissociation constants of 0.8 nM and 2 nM for aFGF and bFGF, respectively. Furthermore, binding of aFGF and bFGF to purified receptor resulted in self-phosphorylation, demonstrating that the isolated receptor had an unaltered intrinsic kinase activity.


Rapid regulation of albumin transcription by insulin and phorbol esters in rat hepatoma cells.

The short-term effects of insulin and phorbol esters on the regulation of the albumin gene in rat H4IIE (H4) hepatoma cells were investigated and compared to the expression of a gene known to be inhibited by these agents, phosphoenol pyruvate carboxykinase (PEPCK). Both insulin and phorbol esters inhibited transcription of the albumin gene in a rapid, dose-dependent manner. Within 15 min, albumin transcription was reduced by approx. 80%. The inhibitory effects of insulin were evident at concentrations of insulin as low as 5.10(-11)M, suggesting that these effects were mediated through insulin-specific pathways. The ability of both phorbol esters and insulin to inhibit albumin transcription suggests that the negative control of this gene is a stable feature in H4 cells. The effect of phorbol esters to mimic insulin action on the albumin gene, and on several other genes in this cell line, implies that a common pathway may be shared by both insulin and phorbol esters.


Regulation of lipoprotein lipase by dibutyryl cAMP, cholera toxin, Hepes and heparin in F1 heart-cell cultures.

Regulation of lipoprotein lipase was studied in mesenchymal rat heart-cell cultures. Treatment of the cultures with dibutyryl cyclic AMP or with cholera toxin resulted in an increase in LPL activity and a comparable increase in LPL mRNA. When the cells were exposed to 100 mM Hepes for 24 h, total enzyme activity rose 2-fold and LPL mRNA increased 2.4-fold. After 72 h, there was a 3-fold increase in LPL mRNA and a 4-fold rise in cellular LPL activity, while medium activity increased 20-fold. Exposure of the cultures to heparin for 24 h resulted in a 3.2-fold increase in total activity and a 36-fold increase in medium activity. This increase was not accompanied by any rise in LPL mRNA. Addition of actinomycin D to control dishes for 24 h resulted in a 33% reduction in LPL mRNA and a 43% reduction in enzyme activity. These values were 71% and 56%, respectively, in Hepes-treated cells, indicating that no stabilization of LPL mRNA occurred under these conditions. It can be concluded that in mesenchymal rat heart-cells in culture cAMP and cholera toxin upregulate lipoprotein lipase at the level of transcription. The increase in LPL activity after 24 h exposure to Hepes could be compatible with transcriptional regulation, while exposure to heparin is not accompanied by a change in LPL mRNA.


The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.


cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.


Identification of an abundant monkey epididymal transcript encoding a homologue of human CAMPATH-1 antigen precursor.

A number of cDNA clones encoding a small (0.5-0.6 kb) transcript have been isolated from a monkey (Macaca fascicularis) epididymal cDNA library. DNA sequence analysis indicates that this abundant epididymal transcript is homologous to the human CAMPATH-1 (CDw52) antigen precursor, a GPI-anchored membrane glycoprotein previously described on lymphocytes and monocytes.


The p60c-src family of protein-tyrosine kinases: structure, regulation, and function.

In 1911, Peyton Rous reported that a fibrosarcoma could be transmitted between chickens in a cell-free extract of the tumor. The transmissible agent, Rous sarcoma virus (RSV), transforms cells by virtue of the presence within its genome of a viral oncogene, v-src, which is derived from a normal cellular gene that has been picked up, or transduced, by the virus. This cellular proto-oncogene, c-src, encodes a protein, p60c-src, which has the ability to phosphorylate proteins on tyrosine residues. Studies of RSV were thus directly responsible for the discovery of cellular proto-oncogenes and of protein-tyrosine kinases, discoveries which have been fundamental in shaping our ideas about cellular growth control. In spite of this, the normal biological role of p60c-src is still unclear and it remains impossible to provide a full answer to the question of how RSV causes tumors. It is clear, however, that c-src is the prototype of a family of at least 8 closely related genes encoding protein tyrosine kinases, the other family members being blk, c-fgr, fyn, hck, lck, lyn, and c-yes. The purpose of this review is to outline our current knowledge of the structure, expression pattern, and function of each of the members of the c-src gene family and to describe recent data which begins to explain how these proteins interact with other cellular proteins to control cell behavior. The evidence for involvement of these proteins in oncogenesis is also discussed.


Adhesion molecules on CD34+ hematopoietic cells in normal human bone marrow and leukemia.

Expression of selected adhesion molecules of the integrin and immunoglobulin family was investigated on CD34+ leukemic cells in 19 AML and 11 ALL cases to evaluate phenotypic differences in adhesive properties of malignant hematopoietic precursor cells in comparison to normal bone marrow CD34+ cells. Of the beta 2-integrin family, CD11a was expressed on > 50% of CD34+ cells in normal bone marrow and almost all leukemias, whereas CD11b and CD11c were not expressed on CD34+ cells in normal bone marrow, but were found on CD34+ blasts in some leukemias of a heterogeneous immunophenotype. Of the beta 1-family, CDw49d (VLA-4) was strongly expressed on normal CD34+ bone marrow cells and on the blasts of all 30 CD34+ leukemic samples, whereas CDw49b (VLA-2) was absent on CD34+ cells in normal bone marrow, but detected on CD34+ cells in a few leukemias which did not constitute a clinical or phenotypic entity according to the FAB classification or immunocytological analysis. The lymphocyte-homing-associated adhesion molecule CD44 (HCAM) and CD58 (LFA-3) were expressed on CD34+ cells in all investigated cases of normal and leukemic bone marrow. ICAM-1 (CD54), the inducible receptor ligand for CD11a/CD18, although present on CD34+ cells in normal bone marrow, was lacking on blast cells of some ALL and AML cases. So far, the variable expression of beta 2-integrins as well as of VLA-2 and of ICAM-1 could indicate distinct differences in cell-cell or cell-matrix adhesion of leukemic cells in ALL and AML patients.


Successful second allogeneic bone marrow transplantation in a relapsed acute myeloid leukemia patient with fungal liver abscess.

Disseminated fungal infection not infrequently complicates the course of allogeneic bone marrow transplantation (allo BMT) in severely immunocompromised patients, and the prognosis of BMT patients who develop systemic fungal infection is very poor. We describe a patient who developed disseminated Candida albicans infection with liver abscess after the first allo BMT for acute myelogenous leukemia (FAB M2). The infection was successfully eradicated by the administration of miconazole and amphotericin B. However, 1 year after the first allo BMT, the patient suffered a relapse of acute myelogenous leukemia with fungal liver abscess. A second allo BMT, accelerating granulocyte recovery by recombinant human granulocyte colony-stimulating factor (rhG-CSF), was successfully performed and the fungal liver abscess resolved with a combination therapy of fluconazole and amphotericin B. The patient is alive and free of both leukemia and fungal disease more than 37 months after the first allo BMT and 25 months after the second allo BMT.


Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.


Prevention of estrus in the queen with chlormadinone acetate administered orally.

The possibility of estrus prevention in the queen by the oral administration of chlormadinone acetate was examined. The animals used were 29 mature and 15 immature queens. For 16 mature females, 4-12.5 mg was given daily by mouth for 7 days every 3 months. Ten of the 16 queens given this treatment came into estrus within 4 months of the first treatment. For 28 females including the immature, 2-12.5 mg was given once a week throughout the experiment. This treatment prevented estrous activity for at least 1 year. In the queens in this study, the side effects were not observed excepting an increase in body weight during treatment. Our results showed that oral administration of this drug weekly is safe and reliable for long-range prevention of estrus in queens.


On the fine structure and complex carbohydrate cytochemistry of the rabbit parotid gland.

The parotid gland of the rabbit, a lagomorph species, was studied by ultrastructural techniques and carbohydrate ultracytochemical stainings. The rabbit parotid gland is a peculiar mixed gland consisting of serous and mucous secretory cells due to their histochemical properties supported by biochemical findings. Acinar cells exhibit heterogeneous features of secretory granules with different electrondensity and occasional presence of subunits. Intercalated duct cells show nuclei with deep indentation and apical granules partly similar to acinar secretory products. Striated ducts are characterized by three different cell populations, namely "light cells" with small secretory granules, "dark cells" rich of scattered mitochondria and typical striated cells. The presence of differentiated cell types within striated duct segments lends credence to the idea that, in addition to the role in electrolyte transport, some ductal cells may be involved in secretion and/or absorption of glycosylated products.


A longitudinal assessment of CSF monoamine metabolite and plasma cortisol concentrations in young rhesus monkeys.

Twenty-two rhesus macaques were studied longitudinally from infancy to early adolescence in order to assess the effects of developmental change, experimental history, gender, and individual variation on the response of the catecholaminergic, serotonergic, and adrenocortical systems to separation-induced stress. Experimental effects were assessed by comparing subjects reared for the first 6 months of life either with their mothers or in peer groups. Developmental changes in response to repeated separation stress were assessed by subjecting the monkeys to four sequential 4-day social separations when they were 6 and 18 months old. At both ages, prior to, and on the last day of the first and fourth separations, cerebrospinal fluid (CSF) was obtained from the cisterna magna to assess monoamine metabolite concentrations, and blood samples were collected to assess plasma cortisol concentrations. Blood samples were also obtained on the first day of each separation at each age. Age-related declines were found in both homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations for all subjects. Social separation consistently increased CSF 3-methoxy-4-hydroxyphenolglycol (MHPG) over baseline levels, but decreased HVA concentrations, whereas 5-HIAA levels increased following the first, but not the fourth separation. Plasma cortisol increased rapidly immediately after separation and remained higher than baseline on day 4 of both the first and fourth separation. Independent of age and experimental condition, peer-rearing increased CSF MHPG and plasma cortisol concentrations. During year 1, peer-rearing also produced diminished CSF 5-HIAA concentrations in female monkeys relative to female mother-reared monkeys, but increased male peer-reared monkeys' concentrations relative to mother-reared males. Interindividual differences were highly stable, with significant correlations both within and between years for each of the three metabolites and cortisol.


The clinical relevance of salivary amylase monitoring in bulimia nervosa.

The aim of this study was to evaluate the clinical relevance of amylase level monitoring as an objective measure in diagnosis and assessment of treatment response in bulimia nervosa. Thirty-three subjects who fulfilled DSM-111-R criteria for bulimia nervosa had serum levels of total and salivary amylase monitored during an 8-week treatment trial. At the beginning of treatment, the average total amylase level was within the upper limits of normal, whereas average salivary amylase levels were abnormally high. During the course of treatment, there was a significant reduction in the average salivary isoenzyme to within the normal range. Significant reductions in amylase levels were recorded in patients with good treatment outcome, but not in those with poor outcome. Amylase levels were not significantly correlated with severity of bulimic symptoms. These results do not justify the use of amylase assays as a routine diagnostic or monitoring test, but isoenzyme monitoring may provide useful clinical information in selected cases.


Biogenic amine metabolites in plasma of drug-naive schizophrenic patients: associations with symptomatology.

The main metabolites of dopamine, noradrenaline, and serotonin, homovanillic acid, methoxyhydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA), were estimated in plasma of 20 drug-naive young male schizophrenic patients and 21 healthy controls. Although there were no significant differences between the two groups, multiple regression analysis revealed strong associations of the patients' 5-HIAA and MHPG plasma levels to their scores in the Brief Psychiatric Rating Scale items. 5-HIAA levels were mainly related positively to hostility and negatively to somatic concern, and MHPG positively to disorientation and negatively to emotional withdrawal and uncooperativeness. The results suggest that levels of the neurotransmitter metabolites may be related to certain psychological dysfunctions of the patients rather than to disease entities.


Proliferative responses of peripheral blood mononuclear cells from patients with autoimmune thyroid diseases to synthetic peptide epitopes of human thyroid peroxidase.

In order to analyze T cell epitopes of human thyroid peroxidase (TPO), 60 peptides based on the sequence of TPO were synthesized and used as antigens in a peripheral blood mononuclear cell (PBMC)-proliferation assay. PBMCs were obtained from 19 patients with Graves' disease, 19 patients with Hashimoto's thyroiditis, and 24 normal subjects. Significant proliferation of PBMC to these peptides occurred only among the autoimmune thyroid disease (AITD) patients, whereas normal subjects did not respond to any of the peptides with a stimulation index over 2. Many peptides induced isolated positive responses and eight produced stimulation of PBMCs from multiple patients on comparison to control PBMC responses. To confirm the significance of reactivity to the peptides PBMCs from four patients were studied on two occasions, and the proliferative responses found to be reproducible. Four peptides, designated according to the amino acid sequence as p110-129, p211-230, p842-861, and p882-901, stimulated patients' PBMCs in a dose-dependent manner. The optimal concentration was 10 micrograms/ml. An anti-HLA-DR monoclonal antibody directed against a monomorphic determinant of the DR molecule was able to block the responses. A significant correlation was found between the PBMC responses to these peptides and responses to microsomal antigen (McAg)/TPO. These data suggest that four peptides corresponding to the amino acid sequences 110-129, 211-230, 842-861 and 882-901 are T cell epitopes of TPO.


The role of the cancer registry in cancer control.

It has been accepted generally that the cancer registry has more of a 'back room' than a 'front line' role in cancer control, its particular responsibilities lying in description of cancer patterns, care, and outcome, in monitoring these variables in relation to control activities, and in providing a research database--often, for others to utilize. While readily justifiable, this prevailing concept of the cancer registry's role may not be sustainable in times of economic restraint. A survey of members of the International Association of Cancer Registries showed that most registries fit the accepted mold. Some, however, extend beyond it, particularly in the direct conduct of epidemiologic research and in the implementation of control programs, particularly screening. Sixteen percent appeared only to be collecting incidence statistics and may be at risk of economic rationalization. It would be consonant with their basic role and skills, and promote more rational cancer control, if cancer registries were to take on an expanded role, including direct participation in epidemiologic research, evaluation of interventions against cancer at the population level, situation analysis and cancer control planning, and implementation of aspects of cancer control--particularly coordination of screening--and monitoring the performance of cancer control programs. This expanded role could become the responsibility of specialized cancer control units of which cancer registration would be the central function.


Gramicidin channel selectivity. Molecular mechanics calculations for formamidinium, guanidinium, and acetamidinium.

Empirical energy function calculations were used to evaluate the effects of minimization on the structure of a gramicidin A channel and to analyze the energies of interaction between three cations (guanidinium, acetamidinium, formamidinium) and the channel as a function of position along the channel axis. The energy minimized model of the gramicidin channel, which was based on the results of Venkatachalam and Urry (1983), has a constriction at the channel entrance. If the channel is not allowed to relax in the presence of the ions (rigid model), there is a large potential energy barrier for all three cations. The barrier varies with cation size and is due to high van der Waals and ion deformation energies. If the channel is minimized in the presence of the ions, the potential energy barrier to formamidinium entry is almost eliminated, but a residual barrier remains for guanidinium and acetamidinium. The residual barrier is primarily due, not to the expansion of the helix, but, to the disruption of hydrogen bonds between the terminal ethanoloamine and the next turn of the helix which occurs when the carbonyls of the outer turn of the helix librate inward toward the ion as it enters the channel. The residual potential energy barriers could be a possible explanation for the measured selectivity of gramicidin for formamidinium over guanidinium. The results of this full-atomic model address the applicability of the size-exclusion concept for the selectivity of the gramicidin channel.


Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion.

The receptor-mediated adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This paper describes a calculational method which simulates the interaction of a single cell with a ligand-coated surface under flow. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the resulting receptor-ligand springs, the response of springs to strain, and the magnitude of the bulk hydrodynamic stresses. The model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the method can generate meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the strain of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same strain. Our analysis of neutrophil adhesive behavior on selectin-coated (CD62-coated) surfaces in viscous shear flow reported by Lawrence and Springer (Lawrence, M.B., and T.A. Springer 1991. Cell. 65:859-874) shows the fractional spring slippage of the CD62-LECAM-1 bond is likely below 0.01. We conclude the unique ability of this selectin bond to cause neutrophil rolling under flow is a result of its unique response to strain. Furthermore, our model can successfully recreate data on neutrophil rolling as function of CD62 surface density.


A semi-empirical approach for the simulation of circular dichroism spectra of gramicidin A in a model membrane.

In an extension of our previous work (Bañó, M. C., Braco, L., and Abad, C. 1991. Biochemistry. 30:886-94), the kinetics of dissociation of gramicidin A double-stranded dimers into beta 6.3-helical monomers in small unilamellar vesicles prepared following different protocols, were investigated using in combination circular dichroism (CD) and high-performance liquid chromatography (HPLC). The analysis of the data from both techniques according to a two-component model strongly supports that any given CD pattern of gramicidin incorporated in the phospholipid bilayer can be deconvoluted essentially as a linear combination of the reference subspectra calculated for the double-stranded dimer and the helical monomer. An HPLC-based, semi-empirical approach is proposed for the simulation of gramicidin CD curves in the model membrane used, and it is shown that the congruence between theoretical and experimental spectra is very satisfactory.


Properties of the mitochondrial peptide-sensitive cationic channel studied in planar bilayers and patches of giant liposomes.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.


Classification of projection images of crystalline arrays of the mitochondrial, voltage-dependent anion-selective channel embedded in aurothioglucose.

Low-dose electron microscopic images have been recorded from membrane crystals of the mitochondrial, voltage-dependent anion-selective channel, embedded in aurothioglucose. There is considerable variation in the high-resolution detail present in correlation averages computed from these images. Correspondence analysis reveals three classes of "control" averages, with main components of variation involving projected size of the pores and density modulations around the pores and in the corners of the unit cells away from the pores. Pretreatments that affect the functional state of the channel also affect the array averages. In particular, there appears to be a general correlation between the expected effector-induced state (i.e., open and closed) and the projected diameter of the channel lumens in the crystalline arrays.


Conformational model for ion permeation in membrane channels: a comparison with multi-ion models and applications to calcium channel permeability.

The permeation properties of ion channels existing in several conductive states were analyzed. Each state was represented by the one-ion model. A special emphasis was placed on features, assumed to be indicative of a multi-ion mode of channel occupancy such as a deviation of concentration dependence of channel conductance from the Michaelis-Menten equation, an anomalous mole fraction effect, a strong voltage dependence of ion block and coupling of unidirectional fluxes (anomalous Ussing flux ratio). The conformational model was shown to have all these properties. The ion permeation through voltage-sensitive calcium channels fulfilled all the characteristics of the model proposed.


23Na and 1H NMR studies on melittin channels activated by tricyclic tranquilizers.

A dynamic 23Na nuclear magnetic resonance (NMR) technique was applied to the exchange system of Na+ ions present inside and outside large unilamellar vesicles at an equivalent concentration. Addition of melittin to phosphatidylcholine vesicles did not induce any detectable Na+ transport across the membrane but subsequent addition of a trace of chlorpromazine or imipramine did induce Na+ transport. Because the formation of a drug-melittin adduct in a solution was detected by 1H NMR, the activation of melittin channels was assumed to originate from the direct interaction of the drug and melittin.


Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.


Prolines are not essential residues in the "barrel-stave" model for ion channels induced by alamethicin analogues.

In the "barrel-stave" model for voltage-gated alamethicin channels in planar lipid bilayers, proline residues, especially Pro14, are assumed to play a significant role. Taking advantage of a previous synthetic alamethicin analogue in which all eight alpha-aminoisobutyric acids were replaced by leucines, two new analogues were prepared in order to test the effects of Pro14 and Pro2 substitutions by alanines. The alpha-helical content of the three analogues in methanol solution remains predominant (between 63 and 80%). Macroscopic conductance experiments show that a high voltage dependence is conserved, although the apparent mean number of monomers forming the channels is significantly reduced when the substitution occurs at position 14. This is confirmed in single-channel experiments which further reveal faster fluctuations for the modified analogues. These results demonstrate that, although prolines, especially Pro14, are favorable residues for alamethicin-like events, they are not absolute prerequisites for the development of highly voltage-dependent multistate conductances.


Ion channels in icosahedral virus: a comparative analysis of the structures and binding sites at their fivefold axes.

An analysis of the crystallographically determined structures of the icosahedral protein coats of Tomato Bushy Stunt Virus, Southern Bean Mosaic Virus, Satellite Tobacco Necrosis Virus, Human Rhinovirus 14 and Mengovirus around their fivefold axes is presented. Accessibilities surfaces, electrostatic energy profile calculations, ion-protein interaction energy calculations, free energy perturbation methods and comparisons with structures of chelating agents are used in this study. It is concluded that the structures built around the viral fivefold axes would be adequate for ion binding and transport. Relative ion preferences are derived for the binding sites, using free energy perturbation methods, which are consistent with the experimental data when available. In the cases where crystallographic studies determined the existence of ions on the fivefold axes, our results indicate that they would correspond to ions in crystallization or purification buffers. The environment of the fivefold axes are rich in polar residues in all icosahedral viral structures whose atomic coordinates are available, including some that are not being analyzed in detail in this work. The fivefold channel-like structures have most of the basic properties expected for real ion channels including a funnel at the entrance, a polar internal environment with frequent alternation of acidic and basic residues, ion binding sites, the capability to induce ion dehydration and ion transit from the external viral surface to the binding sites.


Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data. Altered hydration at the protein-lipid interface could affect conformation, thereby offering a new route by which membrane protein functioning may be modified.


Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.


The impact of single cell voltage clamp on the understanding of the cardiac ventricular action potential.

In this article we review results obtained during the last decade by the single cell voltage clamp technique on cardiac ventricular myocytes, and we re-evaluate the major ionic currents underlying the cardiac action potential. Since its introduction into cardiac electrophysiology in the late seventies this technique has greatly contributed to our knowledge about the role of the transmembrane ionic currents in the heart. Recent findings gained with this method have confirmed that the inward sodium current is responsible not only for the fast depolarization, but, in part, also for the maintenance of the plateau phase of the action potential. The kinetics of the inward calcium current measured by the single cell voltage clamp technique proved to be much faster than was previously thought. In addition, two types of the inward calcium current with different physiological roles have recently been identified. The L-type calcium current plays an important part in maintaining the plateau phase of the action potential and may cause depolarization at less negative potentials. Although the physiological significance of the T-type calcium current is less clear, it appears to be involved in the pacemaker function of cardiac tissues. Single cell voltage clamp experiments have shown that the inward rectifier potassium current is not independent of time, as described earlier, but it helps to terminate the final phase of repolarization, and presumably controls the resting membrane potential. Recent studies with the single cell voltage clamp method have revealed that the delayed rectifier potassium current has, most probably, more than one component and is extensively modulated by neurotransmitters. Its main role is to initiate and terminate cardiac repolarization. This current is of particular importance in regulating rate-dependent repolarizations. The transient outward current, which rapidly activates and inactivates after depolarization, initiates early fast repolarization and may also take part in rate-dependent repolarization. The ionic carriers of this current are most likely potassium and chloride. The use of the single cell voltage clamp technique has led to the discovery of formerly unrecognized currents, like the ATP-dependent potassium current, the sodium activated potassium current and the chloride currents. The application of the new technique has made it possible to focus more attention on currents which were difficult to study previously, such as Na/K pump and Na/Ca exchanger currents.


Perivascular innervation is lost in experimental atherosclerosis.

Nerve fibers, immunohistochemically positive for neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P, form a perivascular network surrounding the carotid arteries of New Zealand White rabbits. Transmission electron microscopy demonstrates that the nerve fibers are primarily located at the adventitial-medial border. Placing a silastic collar around a carotid artery for 14 days, in rabbits fed a diet high in cholesterol, resulted in a focal, intimal thickening in 10 out of 12 rabbits. Contralateral sham-operated arteries showed no intimal thickening. At sites where intimal thickening occurred, there was a disappearance of the perivascular nerve network. The carotid arteries from rabbits that did not respond to the collar and the sham-operated carotid arteries showed an intact and normal perivascular nerve network. In the group of animals which responded to the collar with intimal thickening, there was evidence of a proliferative response proximal to the collar and in this same tissue there was evidence of degeneration of nerve fibers. In conclusion, it has been demonstrated for the first time that, in regions of the carotid artery where intimal thickening occurred, there was an associated degeneration of the perivascular nerve network. The cause of this degeneration and its functional consequences require further investigation.


Dipeptide backbone conformation and antibody recognition of a viral octapeptide epitope.

The peptide YKGTMDSG (Tyr-Lys-Gly-Thr-Met-Asp-Ser-Gly) represents an important antigenic determinant from the glycoprotein G2 of the pathogenic Rift Valley fever virus. By preparing a series of single-residue substitution peptides, the importance to antigenicity of individual residues within this octapeptide has been determined. Here, we investigated a simple and rapid computational analysis to test for correlations between the observed antigenicity of the substitution analogue peptides and the calculated conformational preferences in local regions of the peptides. Conformational energy analyses were carried out on all dipeptide combinations represented in the wild-type octapeptide and in the single-residue substitution analogue peptides. Conformational similarities and differences between wild-type and substitution dipeptide pairs were determined. The results of these computational analyses were then compared with the data on the relative antigenicity of the wild-type octapeptide and the substitution analogues. This comparison revealed a positive correlation. Substitution peptides showing changes in antigenicity possessed significant changes in the calculated backbone conformation relative to wild type in the dipeptides encompassing the residue substitution. Substitution peptides showing no change in antigenicity similarly showed no significant changes in dipeptide conformation. The potential utility of dipeptide conformational energy analyses and this preliminary structure-activity correlation are discussed.


Salt bridge induced changes in the secondary structure of ionic polypeptides.

The carboxylate-containing homopolypeptides poly(L-glutamate) [poly(Glu)] and poly(L-aspartate) [poly(Asp)] were found to form different types of ordered structures in the presence of poly(L-lysine) [poly(Lys)]. Mixing poly(Glu) with poly(Lys) in aqueous solution at neutral pH results in the instantaneous formation of a gel-like precipitate. The secondary structure of the gel precipitate can be best described as intermolecular antiparallel beta-strands, involving the backbone amide groups, as evidenced by the presence of characteristic amide I bands in the ir spectrum at 1684 and 1612 cm-1. Mixing poly(Asp) with poly(Lys) under identical conditions results in the formation of a fine precipitate with a different morphology. Examination of the ir spectrum of the precipitate revealed that unlike poly(Glu), poly(Asp) did not yield any discrete secondary structure upon precipitation with poly(Lys). Addition of solutions containing Ca2+ or Mg2+ to the poly(Glu)/poly(Lys) aggregates resulted in complete dissolution of the gel, with the disappearance of the ir bands characteristic of the intermolecular hydrogen-bonded network. The results demonstrate the importance of salt bridges in establishing strong hydrogen bonds between the backbone amide groups. Reaggregation occurred upon heating the poly(Glu)/poly(Lys) mixture in the presence of Ca2+, but not in the presence of Mg2+ ions. In the presence of Ca2+ ions, aggregation and formation of an extended hydrogen-bonded network occurred upon heating. The aggregates formed upon heating poly(Glu)/poly(Lys) in the presence of Ca2+ were attributed solely to complexation of Ca2+ to the carboxylate groups of poly(Glu) with poly(Lys) remaining free in solution. Dissolution of the aggregate could be accomplished through addition of Mg2+ at room temperature.


Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.