Localization of heterogeneous nuclear ribonucleoprotein in the interphase nuclear matrix core filaments and on perichromosomal filaments at mitosis.

Although heterogeneous nuclear RNA (hnRNA) has been localized to the core filament substructure of the nuclear matrix, its precise location in the filament network has been unknown. The fA12 monoclonal antibody can localize, at high resolution, hn ribonucleoproteins (hnRNPs) and, presumably, hnRNA. Gold bead immunolabeling of resinless electron microscopy sections showed the fA12 antigens were in the fibrogranular material enmeshed in the filament network and not in the filaments themselves. At mitosis, hnRNP antigens became dispersed into a halo surrounding the chromosomes and spindle poles. Immunogold staining showed fA12 stained fibrogranular material associated with perichromosomal and pericentriolar filaments distinct from the mitotic spindle fibers. fA12 also labeled the midbody remaining after cytokinesis.     

Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy.

OBJECTIVE: To determine the frequency of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy. DESIGN: Mutation screening of the terminal 200 base pairs of connexin43 gene coding sequence in a series of patients from tertiary care centres. PATIENTS: 48 patients with visceroatrial heterotaxy attending UK Regional Paediatric Cardiology Centres. RESULTS: No changes from the published connexin43 consensus sequence were found in any of the 48 patients studied. CONCLUSIONS: Germline mutations of the phosphorylation sites in teh regulatory domain of the connexin43 gene are rare in patients with visceroatrial heterotaxy.     

Marking of resection margins.

Starch was used to mark the resection margins of breast tissue simply by rolling formalin fixed specimens in, for instance, glove powder. Starch adheres satisfactorily to the specimen and is obvious, microscopically, if crossed polarisers are used. There is little "carry-over" of starch across the rest of the tissue, and subsequent radiology of specimen or blocks is not prejudiced. It is concluded that starch powder is eminently suitable in most cases as a single marker of the surgically cut surface. The method is quick, cheap, and clean. It should not be relied on, anymore than other methods, to mark the surgically cut surface of ragged or partly disrupted specimens.     

Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with beta-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine.     

Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells

We have developed a simple yet powerful technique for delineating the morphological events of tumor-induced angiogenesis and other tumor-induced host processes with dual-color fluorescence. The method clearly images implanted tumors and adjacent stroma, distinguishing unambiguously the host and tumor-specific components of the malignancy. The dual-color fluorescence imaging is effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice. This model shows with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes. The GFP-expressing tumor vasculature, both nascent and mature, could be readily distinguished interacting with the RFP-expressing tumor cells. GFP-expressing dendritic cells were observed contacting RFP-expressing tumor cells with their dendrites. GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells. GFP lymphocytes were seen surrounding cells of the RFP tumor, which eventually regressed. Dual-color fluorescence imaging visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations.