Biomedical subjects

M Jarnik

Publications and source records attributed to M Jarnik.

13 recordsLinked to original sources

PubMed2000

Ama1p is a meiosis-specific regulator of the anaphase promoting complex/cyclosome in yeast.

Meiosis is the developmental program by which diploid organisms produce haploid gametes capable of sexual reproduction. Here we describe the yeast gene AMA1, a new member of the Cdc20 protein family that regulates the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). AMA1 is developmentally regulated in that its transcription and splicing occur only in meiotic cells. The meiosis-specific processing of AMA1 mRNA depends on the previously described MER1 splicing factor. Several results indicate that Ama1p is required for APC/C function during meiosis. First, coimmunoprecipitation assays indicate that Ama1p associates with the APC/C in vivo. Second, Ama1p is required for the degradation of the B-type cyclin Clb1p, an APC/C substrate in both meiotic and mitotic cells. Third, ectopic overexpression of AMA1 is able to stimulate ubiquitination of Clb1p in vitro and degradation of Clb1p in vivo. Mutants lacking AMA1 revealed that it is required for the first meiotic division but not the mitotic-like meiosis II. In addition, ama1 mutants are defective for both spore wall assembly and the expression of late meiotic genes. In conclusion, this study indicates that Ama1p directs a meiotic APC/C that functions solely outside mitotic cell division. The requirement of Ama1p only for meiosis I and spore morphogenesis suggests a function for APC/C(Ama1) specifically adapted to germ cell development.

Amino Acid Sequence↗

PubMed2000

Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.

Adaptation, Biological↗

PubMed2000

Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma.

Mutations in the cornified cell envelope protein loricrin have been reported recently in some patients with Vohwinkel syndrome (VS) and progressive symmetric erythrokeratoderma (PSEK). To establish a causative relationship between loricrin mutations and these diseases, we have generated transgenic mice expressing a COOH-terminal truncated form of loricrin that is similar to the protein expressed in VS and PSEK patients. At birth, transgenic mice (ML.VS) exhibited erythrokeratoderma with an epidermal barrier dysfunction. 4 d after birth, high-expressing transgenic animals showed a generalized scaling of the skin, as well as a constricting band encircling the tail and, by day 7, a thickening of the footpads. Histologically, ML. VS transgenic mice also showed retention of nuclei in the stratum corneum, a characteristic feature of VS and PSEK. Immunofluorescence and immunoelectron microscopy showed the mutant loricrin protein in the nucleus and cytoplasm of epidermal keratinocytes, but did not detect the protein in the cornified cell envelope. Transfection experiments indicated that the COOH-terminal domain of the mutant loricrin contains a nuclear localization signal. To determine whether the ML.VS phenotype resulted from dominant-negative interference of the transgene with endogenous loricrin, we mated the ML.VS transgenics with loricrin knockout mice. A severe phenotype was observed in mice that lacked expression of wild-type loricrin. Since loricrin knockout mice are largely asymptomatic (Koch, P.K., P. A. de Viragh, E. Scharer, D. Bundman, M.A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, et al., J. Cell Biol. 151:389-400, this issue), this phenotype may be attributed to expression of the mutant form of loricrin. Thus, deposition of the mutant protein in the nucleus appears to interfere with late stages of epidermal differentiation, resulting in a VS-like phenotype.

Amino Acid Sequence↗

PubMed1998

Genetic structure of the ancestral population of modern humans.

Neutral DNA polymorphisms from an 8-kb segment of the dystrophin gene, previously ascertained in a worldwide sample (n = 250 chromosomes), were used to characterize the population ancestral to the present-day human groups. The ancestral state of each polymorphic site was determined by comparing human variants with their orthologous sites in the great apes. The "age before fixation" of the underlying mutations was estimated from the frequencies of the new alleles and analyzed in the context of these polymorphisms' distribution among 13 populations from Africa, Europe, Asia, New Guinea, and the Americas (n = 860 chromosomes in total). Seventeen polymorphisms older tan 100,000-200,000 years, which contributed approximately 90% to the overall nucleotide diversity, were common to all human groups. Polymorphisms endemic to human groups or continentally restricted were younger than 100,000-200,000 years. Africans (six populations) with 13 such sites stood out from the rest of the world (seven populations), where only 2 population-specific variants were observed. The similarity of the frequencies of the old polymorphisms in Africans and non-Africans suggested a similar profile of genetic variability in the population before the modern human's divergence. This ancestral population was characterized by an effective size of about 10,000 as estimated from the nucleotide diversity; this size may describe the number of breeding individuals over a long time during the Middle Pleistocene or reflect a speciation bottleneck from an initially larger population at the end of this period.

Alleles↗

PubMed1998

Cornified cell envelope assembly: a model based on electron microscopic determinations of thickness and projected density.

In stratifying squamous epithelia, the cornified cell envelope (CE), a peripheral layer of crosslinked protein, is assembled sequentially from precursor proteins initially dispersed in the cytoplasm. Its major component is loricrin (37 kDa in mouse), which contributes from approx. 60% to >80% of the protein mass in different tissues. Despite its importance to the mechanical resilience and impenetrability of these tissues, detailed information has not been obtained on CE structure, even on such basic properties as its thickness or uniformity across a given CE or from tissue to tissue. To address this issue, we have studied CEs isolated from three murine epithelia, namely epidermis, forestomach and footpad, by electron microscopy of metal-shadowed specimens and scanning transmission electron microscopy (STEM) of unstained specimens. The former data reveal that the cytoplasmic surface is smoothly textured whereas the extracellular surface is corrugated, and that the average thickness is 15.3+/-1.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.0+/-0.8 kDa/nm2, which were remarkably consistent over all three tissues. These data imply that the mature CE has a uniquely defined thickness. To explain its uniformity, we postulate that loricrin forms a molecular monolayer, not a variable number of multiple layers. In this scenario, the packing density is one loricrin monomer per 7 nm2, and loricrin should have an elongated shape, 2.5-3.0 nm wide by approx. 11 nm long. Moreover, we anticipate that any inter-tissue variations in the mechanical properties of CEs should depend more on protein composition and cross-linking pattern than on the thickness of the protein layer deposited.

Animals↗

PubMed1997

Nuclear DNA diversity in worldwide distributed human populations.

Nucleotide variation was examined in an 8 kb intronic DNA bordering exon 44 of the human dystrophin gene on Xp21. Thirty-six polymorphisms (substitutions, small insertions/deletions and one (T)n microsatellite) were found using SSCP/heteroduplex analysis of DNA samples from mixed Europeans, Papua New Guineans as well as from six African, three Asian and two Amerindian populations. In this way the European bias in the nuclear polymorphism ascertainment has been avoided. In a maximum likelihood tree constructed from the frequency data, Africans clustered separately from the non-African populations. Fifteen polymorphisms were shared among most of the populations compared, whereas 13 sites were found to be endemic to Africans and four to non-Africans. The common sites contributed most to the average heterozygosity (Hn=0.101%+/-0.023), whereas the endemic ones, being rare, had little effect on this estimate. The F(ST) values were lower for Africans (0.072) than for non-Africans (0.158), suggesting a higher level of gene exchange within Africa, corroborating the observation of a greater number of segregating sites on this continent than elsewhere. The data suggest a recent common origin of the African and non-African populations, where a greater geographical isolation of the latter resulted in a smaller number of newly acquired polymorphisms.

Cell Nucleus↗

PubMed1997

Spatial distributions of sulfur-rich proteins in cornifying epithelia.

We have used energy-filtered electron microscopy and electron energy loss spectroscopy (EELS) to characterize the distributions of sulfur-rich proteins in granular layer cells of squamous stratifying epithelia and their redistribution in the cornified layer, with particular attention to assembly of the cornified cell envelope (CE). Our measurements provide quantitative information that complements highly specific but qualitative data from immunocytochemistry. Spatial distributions of sulfur, phosphorus, and nitrogen were mapped in unstained thin sections of mouse epidermis and forestomach, using a postcolumn energy filter. Nitrogen images were indicative of total protein while phosphorus images provided a control to validate the algorithms used to calculate the elemental maps. Sulfur was found at high levels in round L-granules in the granulocyte cytoplasm and in the cornified CE, correlating with the presence of the protein, loricrin ( approximately 7% Cys/Met residues). EELS confirmed these observations quantitatively: either L-granules consist exclusively of loricrin or any additional components must have an equally high net sulfur content. These data also confirm the large fraction (approximately 75%) of loricrin in the CE, as inferred from modeling of its amino acid composition. We also observed extracellular deposits between cornified squames in fetal mouse epidermis that we call peripheral granules. Their sulfur content is at least as high as that of L-granules but they do not label with anti-loricrin antibodies, suggesting the presence of another sulfur-rich protein.

Animals↗

PubMed1996

Overall informativity, OI, in DNA polymorphisms revealed by inter-Alu PCR: detection of genomic rearrangements.

We studied two systems of multilocus markers revealed by PCR using primers directing amplification between Alu repeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-Alu PCR products. A great proportion of inter-Alu PCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OI of a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). High OI values (5.8 and 11.5) of the two presented systems of inter-Alu PCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22-qter region in sporadic colon cancer.

Animals↗

PubMed1996

Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia.

In the final stages of terminal differentiation in the epidermis and other squamous epithelia, a approximately 15 nm thick protein layer called the cornified cell envelope (CE) assembles on the keratinocytes' inner surface. Its constituent proteins are covalently crosslinked by the action of transglutaminases. Recent studies have indicated that the expression of CE precursor proteins may vary in different tissues. To investigate such variations further, we have studied the CEs of two different keratinizing epithelia of mouse: epidermis and forestomach, with particular focus on their contents of loricrin and the small proline-rich proteins (SPRs). To this end, we have applied electron microscopic immunocytochemistry and estimated the CE protein compositions by mathematical modeling of their amino acid compositions. Ultrastructurally, forestomach resembles the epidermis in having well defined cornified and granular layers. Minor but significant differences are: in forestomach, striated material resembling lamellar granules is intercalated between the cornified squames; and in forestomach granular layer cells, loricrin-containing L-granules are more abundant, and filaggrin-containing F-granules less abundant than in epidermis. In forestomach, dense labeling with anti-SPR1 antibody was observed at the margin of cornified layer cells; and in the granular layer, diffuse but positive labeling of both cytoplasm and nucleus. In contrast, epidermis was uniformly negative. Isolated forestomach CEs (but not epidermal CEs), labeled positively on the cytoplasmic side, consistent with the presence of covalently crosslinked SPR1. Our compositional analysis predicts the content of loricrin in forestomach CEs to be very high (approximately 65%), as in the epidermis, and accompanied by approximately 18% content of total SPRs. Of these, a substantial proportion should be SPR1, according to our immunolabeling data. In contrast, epidermal CEs are calculated to have a much lower amount of SPRs or SPR-like proteins (approximately 8%), with a negligible content of SPR1. Thus both kinds of CEs have loricrin as their major constituent but differ in their respective complements of SPRs, which are thought to inter-connect loricrin molecules in the final phase of CE assembly. Applying a basic concept of materials science, it may be that the observed differences in their SPR contents reflect differences in the mechanical and chemical properties required for the function of the respective CEs.

Amino Acid Sequence↗

PubMed1995

Alu-PCR combined with non-Alu primers reveals multiple polymorphic loci.

A marker suitable for genetic mapping and genomic fingerprinting is characterized by high polymorphic information content (PIC) and high "multiplex ratio" (defined as the number of loci that can be simultaneously typed). Towards this goal, we combined an Alu-specific with a non-Alu primer in a single PCR amplification targeting genomic regions where length polymorphisms are abundant. Three loci were revealed with the variable number of (AAT), (TAAA), (AG), and/or (AAAGG) motifs, and PIC values between 0.7 and > 0.94. Their location on Chromosomes (Chrs) 19q12, 17q12-q24, and 5q31.2-33.3 was determined by multipoint analysis with markers from CEPH database. The most common genotype for this three-locus marker, estimated from the occurrence of the most frequent alleles, is of the order of 2 x 10(-4), while the combined PIC value of a single typing experiment is 2.37. The use of a similar primer pair, as well as examples from the literature, indicates the general nature of this approach when a non-Alu oligonucleotide, presumably with "random" priming sites downstream of Alu repeats, is combined with an Alu-specific one. Clustering of DNA length variants in the regions adjacent to interspersed repeats provides opportunity to develop other highly informative multiple-locus markers similar to that described here.

Alleles↗

PubMed1991

Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells.

In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minutes. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.

Animals↗

PubMed1991

Toward a more complete 3-D structure of the nuclear pore complex.

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.

Actins↗

PubMed1990

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

Animals↗