Structure of intermediate filament assembly in hair deduced from hydration studies using small-angle neutron scattering.

Intermediate filaments (IFs) are ubiquitous in biological structures including hair. Small-angle neutron scattering (SANS) data from hydrated samples were used in this study to investigate the distribution of water in hair, and model the structure of the IF assembly. A main diffraction peak at a d-spacing of ∼90 Å, and two weaker reflections show that IFs are arranged in a ∼105 Šquasi-hexagonal lattice. Changes in the diffraction peaks show that only a small fraction of the water absorbed by hair enters between the IFs, and little water diffuses into the core of the IFs. The amount of water in the IF assembly increases rapidly up to 10% relative humidity (RH), and then slowly with further increase in RH. Most of the water appears to reside outside the IF assembly, in the voids and at the interfaces, and contribute to the central diffuse scattering. The IF assembly in the decuticled hair absorbs more water and is more ordered than that the native hair. This suggests that cuticle acts as a barrier, and might constrain the structure by compressing the cortex radially. Treatments with oils that are hydrophobic, heat treatment, and reduction of the S-S linkages that opens up the matrix by disulfide bond cleavage, all affect structure and water permeability. Coconut oil was found to impede hydration more than the soybean oil because of its ability to penetrate deeper into hair. A new model for the IF assembly that is sterically more favorable than the previous models is proposed.

Case of mistaken identity: bullous congenital ichthyosiform erythroderma mistaken as epidermolysis bullosa simplex.

Distinguishing clinically similar dermatologic disorders can be challenging and the differential diagnosis of a blistering eruption in the newborn period can be extensive. Several genodermatosis, such as bullous congenital ichthyosiform erythroderma (BCIE) and epidermolysis bullosa simplex (EBS), autoimmune bullous disorders, infectious diseases, sucking blisters, and bullous mastocytosis must be considered. We present a case of BCIE misdiagnosed as EBS and review characteristic clinical and histopathological features of each disorder as well as the basic approach to treatment.

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy.

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.

Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs).

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Pseudoneoplastic proliferation of histiocytes with paclitaxel-induced ultrastructural changes in a mastectomy specimen.

A 49-year-old Hispanic woman with a T4N1M0 infiltrating duct carcinoma of the left breast underwent four courses of FAC (doxorubicin 86 mg, 5-fluorouracil 860 mg, cyclophosphamide 86 mg, and dexamethasone 10 mg) adjuvant chemotherapy plus four courses of paclitaxel (Taxol; Bristol-Myers Squibb Oncology, Princeton, NJ) and subsequent mastectomy. The tumor shrunk from 6.5 cm to 2.5 cm after the treatment. The residual tumor in the surgical specimen measured 1.5 cm with eight positive out of 24 axillary lymph nodes. The tumor showed typical chemotherapy changes and a massive proliferation of histiocytes that mimicked a neoplasm. A nodular proliferation of the same cells in one axillary node raised the impression of a second malignant tumor in the breast spreading to the node. The histiocytic cells contained lamellar and coarse periodic acid-Schiff-positive material distending their cytoplasm and they were strongly positive for CD68 and negative for CD1a, pan keratin, and S-100. These findings ruled out histiocytoid carcinoma, granular cell tumor, and Erdheim-Chester disease. The proliferating histiocytes had ultrastructural findings of paclitaxel-induced cytotoxicity with disorganized stacks of intermediate filaments positive for vimentin by immunostains and fewer masses of tubulin. The treated breast carcinoma cells were tubulin-positive but the proliferating histiocytes were tubulin-negative.

Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules.

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.

Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis).

Neuropile glial (NPG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis, were studied by histological, histochemical and immunocytochemical techniques. The NPG cells are often surrounded by electron-dense microglial cells. The central cytoplasm of NPG cells shows a significant zonation. The zone around the nucleus contains mitochondria, glycogen and vesicles. The cytoplasm also contains many ribosomes, a few dictyosomes and distinct inclusions up to 2 microns in diameter. A second zone around the perinuclear region is marked by the occurrence of bundles of intermediate filaments that correspond in thickness to glial filaments of vertebrates. We found a positive reaction with polyclonal antibodies against human glial fibrillary acidic protein (GFAP), and the areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid. Therefore, the membrane stacks could be part of an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism.

Induction of aggregation and augmentation of protein kinase-mediated phosphorylation of purified vimentin intermediate filaments by withangulatin A.

Purified assembly-competent vimentin, an intermediate filament protein, was obtained from bovine lens in this study. The effects of withangulatin A on vimentin assembly with or without phosphorylation were examined by negative-stain electron microscopy. Soluble tetrameric vimentin was assembled into irregular fibrils with lateral associations or short filaments after pretreatment with 50 or 100 microM withangulatin A, respectively. Incubation of assembled vimentin filaments with withangulatin A at 50 or 100 microM resulted in the formation of aggregates, and the degree of aggregation was concentration dependent. The appearance of vimentin filaments was slightly altered after treatment with cAMP-dependent protein kinase or protein kinase C; however, phosphorylation of filamentous vimentin by the protein kinases in the presence of withangulatin A resulted in higher degrees of aggregation of the filaments, compared with those treated only with the drug. Moreover, the level of phosphorylation of filamentous vimentin by the protein kinases was augmented in the presence of withangulatin A. Experimental results indicated that withangulatin A directly and specifically affects the conformation of the vimentin molecules, thereby resulting in alterations in assembly behavior and reactivity toward cAMP-dependent protein kinase and protein kinase C. The data observed further imply that withangulatin A, which directly causes aggregation of vimentin filaments, is a vimentin intermediate filament-targeting drug.

Purification and immunological detection of pea nuclear intermediate filaments: evidence for plant nuclear lamins.

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.

Hyperbaric oxygen accelerates the neurotoxicity of 2,5-hexanedione.

The molecular pathogenesis of n-hexane neurotoxicity has been postulated to proceed as follows: The gamma-diketone metabolite, 2,5-hexanedione (HD), reacts with lysyl-amino groups on neurofilaments to form imines. The imines cyclize to form pyrroles. The pyrroles autoxidize, resulting in covalent protein-protein crosslinking within or between neurofilaments. A resultant impairment of neurofilament transport is proposed to lead to neurofilament-filled axonal swellings. This experiment was designed to test whether oxidation is a necessary pathogenetic step in vivo by comparing time of onset of paralysis of an HD treated group of rats to that of a group receiving HD plus oxygen under high pressure (OHP). The group of rats receiving the hyperbaric oxygen treatment reached the endpoint of hindlimb paralysis significantly sooner than the group receiving none. The fact that OHP does accelerate HD neuropathy points towards an oxidative step in the molecular pathogenesis of gamma-diketone neuropathy.

Selective growth in culture of fetal rat renal collecting duct anlagen. Morphologic and biochemical characterization.

Cell culture conditions were devised that selectively supported growth of 13 or 14 gestation day F344 rat ureteric bud, the renal collecting duct anlagen. These same conditions also inhibited the growth of metanephrogenic mesenchyme, precursor of structures proximal to the duct. Isolated buds were cultured in Ham's F12 medium supplemented with epidermal growth factor, selenium, insulin, hydrocortisone, prostaglandin E1, transferrin, and triiodothyronine; fetal bovine serum (1%) was required for continuous propagation. Cultured cells were epithelial in morphology and formed domes. By electron microscopy, many structural characteristics of highly differentiated cells were evident: numerous mitochondria, Golgi apparatus, extensive endoplasmic reticulum, an occasional cilium, intracytoplasmic filaments, polarized formation of microvilli, and gap junctions. Histochemistry revealed considerable functional differentiation as well. Cultured bud cells, adult collecting duct, and fetal duct anlagen were positive for acid phosphatase, membrane-localized ATPase, and nonspecific esterase. Bud cells and fetal duct anlagen expressed high levels of gamma-glutamyl transpeptidase activity while adult collecting duct exhibited slight activity. In addition, immunocytochemical observation of intermediate filament expression revealed the presence of epithelial cytokeratins but absence of mesenchymal vimentin in cultured bud cells and fetal and adult collecting ducts. These results indicate that the culture conditions described can maintain the partially differentiated fetal collecting duct anlagen in a state consistent with its embryonal derivation, and therefore may be useful in culture studies of renal differentiation.