It is well known that cephalopods ( octopus, squid, cuttle fish etc. ) have various pulsatile systems in their bodies. We have now used the gills of Loliolus investigatoris and Loligo dauvauceli, two small squids found in the Bay of Bengal, as a system for studying such autonomous pulsations. The gills excised out of squids cast on the beach from fishing nets can be maintained in pasteurised sea water for 3 4 or 5 days in petri dish where they continue to execute the rhythmic movements.

The gill samples were fixed in 2% Gluteraldehyde in 0.1M Phosphate buffer (pH 7.4).for 48 hours, then washed with filtered pasteurised sea water, followed by washing in distilled water, dehydration in acetone and critical point drying in liquid CO2 and coating with gold.

Fig. 1.depicts an ablated gill which executes an oscillatory motion as a whole; more importantly, the individual finger like projections shown in fig. 2, exhibit rhythmic lateral movements and the blood-vessels, as a result of constrictions and dilation, seem to “tick” like a clock in rapid succession. Periodicity of the gill swinging as a whole is about one swing per 7-8 seconds, for the sidewise swing it varied from 35 to 70 seconds in one gill, 11-39 in another while the “ticking” is about once a second. This last one is, however, lost after a few hours.

Ncd is a kinesin-related microtubule motor protein of Drosophila that plays essential roles in spindle assembly and function during meiosis in oocytes and mitosis in early embryos. Antibody staining experiments have localized the Ned motor protein to spindle fibers and spindle poles throughout the meiotic and early mitotic divisions, demonstrating that Ncd is a spindle motor.

We have made ncd-gfp gene fusions with wild-type and S65T gfp and expressed the chimaeric genes in Drosophila to target GFP to the spindle. Transgenic Drosophila carrying the ncd-gfp gene fusions in an ncd null mutant background are wild type with respect to chromosome segregation, indicating that the Ncd-GFP fusion proteins can replace the function of wild-type Ncd. The Ncd-GFP fusion proteins in transgenic Drosophila are expressed under the regulation of the native ncd promoter.

Analysis of live Drosophila oocytes and early embryos shows green fluorescent spindles, demonstrating association of Ncd-GFP with meiotic and mitotic spindles. In mitotic spindles, Ncd-GFP localizes to centrosomes (Fig. 1a) and spindle fibers (Fig. 1b).

Cell division is thought to be powered by the constriction of an actomyosin containing contractile ring found transiently in the cleavage furrow. Conventional myosin II plays a fundamental role in this process of cytokinesis where, in the form of a multimeric complex known as the bipolar thick filament, it is thought to be the molecular motor that generates the force necessary to cause ring constriction.

In order to study the dynamics of this protein in the dividing cell, we have made a fusion protein of the green fluorescent protein (GFP) and the amino terminus of the Dictyostelium myosin heavy chain (GFP-myosin), and imaged the location of this protein in dividing Dictyostelium cells were it is the only myosin II present in the cell. The addition of GFP does not compromise the functioning of the myosin motor as evidenced by the fact that purified GFP-myosin has solution ATPase and in vitro motility kinetics similar to that of non-labelled myosin. In addition, GFP-myosin fully complements the myosin null mutation for both development and cytokinesis in suspension suggesting that GFP-myosin acts as a regulated motor when expressed in cells.

As ligand-regulated transcription factors, glucocorticoid receptors (GR) must traffic through the cytoplasm, traverse the nuclear pores, and subsequently traffic within the the nucleus to reach their target genes. Due to technical difficulties with immunocytology, little is known about the translocation process or the intranuclear localization. The recent characterization of a chromophore, green fluorescent protein (GFP), provided a general tool to fluorescently label proteins in living cells. With the development of a transcriptionally active GFP-GR chimera, it became possible to visualize GR translocation and intranuclear distribution in living cells.

This chimeric receptor was transiently transfected into mouse adenocarcinoma cells, allowing the direct visualization of GR using real-time video and confocal laser scanning microscopy. Mobility of GFP-GR was analyzed with fluorescent recovery after photobleaching (FRAP).

The hormone-free GFP-GR was localized in the cytoplasm figure 1). Dexamethasone (lOnM) initiated GFP-GR translocation into the nucleus (Figure 2 and 3). The translocation rate was dose- and temperature-dependent, and occurred in a pulsatile manner along cytoplasmic fibrillar structures (Figure 2). FRAP experiments showed that GFP-GR remained in motion within the nucleus after translocation.

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

Apoptosis or programmed cell death, is a mechanism of cellular demise which is believed to play a role in many physiological processes, and which when defective, can contribute to the pathogenesis of cancer. Apoptosis is stimulated in cells by the p53 tumor suppresser, Bax and El A proteins and inhibited by overexpression of Bcl-2 and adenovirus E1B 19 kDa oncoproteins. A role for altered apoptosis in cancer comes from recent reports which have documented that inactivation of p53 (which promotes apoptosis) and/or overexpression of bcl-2 (which inhibits apoptosis) occurs in numerous cancers. Recent work from our laboratory has shown that inactivation of p53 (either by physical complex formation between p53 and Human Papillomavirus (HPV) type 16- or 18- E6 protein or by mutation of p53), is associated with increased expression of bcl-2 in cervical carcinoma cells. The combination of inactive p53 and increased expression of bcl-2 may lead to inhibition of apoptosis and thereby contribute to the development or progression of cervical cancer.

Previous studies have demonstrated that GTPgS and rab5:Q79L, a constitutively active rab5 mutant, stimulate endosome fusion in in vitro assays [Cell Reg. 1:113-124 (1989)]. Additionally, electron microscopy has shown that under these conditions, endosomes form incompletely fused couplets [Cell Reg. 1:113-124 (1989); Arch. Biochem. Biophys. 326:64-72 (1996)]. CHO and BHK cells that overexpress rab5:Q79L develop ‘giant’ cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab 5 and transferrin receptor (data not shown). The formation of these vesicles has been analyzed by time-lapse video microscopy which shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These endosome fusion events occur by two distinct mechanisms that differ in temporal and spatial characteristics of the membrane merger process and we refer to these processes as “explosive” versus “bridge” fusion. “Explosive” fusion is a rapid process in which the initial opening that forms between contacting vesicles expands rapidly and permits an abrupt, complete coalescence of membranes (Fig. 1).

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

Detection of integrated HPV DNA in SiHa and CaSki cells has been demonstrated by in situ PCR (1,2,3) and more recently TSA (4). These reports focused on high sensitivity single copy HPV detection using chromogenic techniques. Here we utilize high resolution and high sensitivity fluorescent methods of detection to analyze the spatial relationship of HPV DNA with respect to subnuclear domains enriched in splicing-related proteins, shown here by immunofluorescent detection of the SC-35 antigen (5). In situhybridization was performed using conventional reagents and the resulting probe signal amplified using fluorescent labeled tyramides (6). In contrast to techniques such as in situ PCR which require specialized procedures and equipment, detection of HPV DNA in SiHa cells was rapidly and routinely achieved using no specialized equipment. In addition, high target resolution was maintained, in contrast to in situ PCR, which is plagued by signal dispersion and difficult localization (7).

Recently, spectral imaging has been successfully applied to 24-color fluorescence in situ hybridization (FISH), and it has evolved into a new powerful method for the analysis of structural and numerical chromosomal aberrations on metaphase spreads (spectral karyotyping) For cytological specimens it is often impossible to use fluorescence microscopy because of (fixation-induced) auto-fluorescence or prior cytological staining. Also, re-examination of archived fluorescent specimens is hampered by fading. However, the need for multi-parameter cytochemical analysis remains when rare or unique material is to be studied in research or clinical diagnosis. Multi-color bright-field microscopy using enzyme precipitates has become feasible for in situ hybridization as well as immunohistochemical detection. Conventional transmission microscopy optics have allowed up to three targets to be detected simultaneously. In parallel to 24-color FISH, we explored the potential of spectral imaging to increase the number of parameters to be analyzed in bright-field microscopy.

We describe the easy and rapid detection of oxytocin oligonucleotide probes in in situ hybridization using a biotinyl tyramide amplification system (TSA).

There are several some instances where related proteins have sequence homology so great that only oligonucleotide probes will allow one to distinguish between their mRNAs. Vasopressin and oxytocin are such a pair. Traditional in situ hybridizationusing 35S labeled probes allows the specific detection of oxytocin mRNA.See FIG. 1.

Nonradiometnc detection of oligonucleotide probes can be very difficult since the amount of labeling possible is limited by the small size of the probe. Traditional nonradiometric detection of a 30-mer biotin-labeled oxytocin probe with streptavidin-HRP followed by DAB detection proved essentially negligible. See FIG. 2.

A novel tyramide amplification system (TSA) was used to increase the detection of the oxytocin probe signal. Most of the protocol was unchanged from the standard nonradioactive methodology. The biotin-labeled probe was hybridized overnight as usual. Stringency washes were also done as usual the next morning.

Deregulation of epidermal growth factor receptor (EGFR) gene expression is belived to be a major factor in the development of epithelial cell tumorigenesis, including hepatic carcinomas. To examine EGFR expression within the course of liver cirrhosis, we have identified its hepatic mRNA distribution using a biotinylated antisense oligonucleotide probe.

In this study liver samples from liver transplantation recipients were fixed with 4% formaldehyde in PBS for 24 h and embedded in paraffin. Following treatments aimed to promote probe penetration and eliminate non-specific probe binding, an EGFR antisense biotin-labeled oligonucleotide probe was hybridized to sections at 45°C for 4h. High and low stringency washes were designed to remove the unbound probe. Following the blocking of non-specific tissue binding, sections were incubated with streptavidin-peroxidase. Biotinylated tyramide was then used for signal amplification. After washing, streptavidin-peroxidase was reapplied, followed by colorimetric detection of peroxidase activity using DAB as substrate. The sections were counterstained with hematoxylin and coverslipped.

p53 is a tumor suppressor gene which involves apoptosis and cell cycle arrest under certain stress stimulate. The expression of p53 is associated with ischemia-reperfussion induced myocardial apoptosis in rat and with the development of coronary artery restenosis after angioplasty in humans. However, the status of p53 in human cardiomyocytes and fibroblasts in congestive heart failure (CHF) remains controversial.

Transforming growth factor-beta (TGF-β) is a growth-regulating peptide that has been shown to enhance collagen production both in vivo and in vitro in cultured cells. Both p53 and TGF-β may inhibit the cell growth and induce cellular apoptosis. While the previous studies demonstrated that TGF-β1 is present in the animal myocardium, the presentation and localization of TGF-β1 in human myocardium remain unclear.

Therefore, the present study was designed to investigate the p53 and TGF-β1 in human ventricular myocardium in normal subjects and in patients with heart failure. Five normal subjects and five end-stage failing human ventricular myocardium tissue were obtained from cardiac transplantation.

Interferon beta (IFN β) is a member of the family of secreted proteins that compose the host defense against viral, bacterial, and parasitic infections and tumors. The interferons exert their activity indirectly by regulating a number of other proteins. Low level expression of IFN β has been reported in a variety of non-epithelial cells from normal animals. Here, we demonstrate the expression of IFN β in epithelial cells lining organs that are chronically exposed to a variety of environmental challenges.

Tissue samples from the digestive system, urinary bladder, mammary fat pad, skin and cornea of C3H/HeN mice were fixed with 10% formalin and processed for histology. Immunoperoxidase was performed on tissue sections that had been de-waxed and treated with trypsin. Briefly, the sections were incubated with rabbit anti mouse IFN β (Lee Biomolecular, San Diego, CA) overnight at 4°C, washed and incubated further with a peroxidase conjugated goat anti-rabbit IgG, (F(ab) 2) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at ambient temperature.

Immunolocalizaton studies of type VI collagen in skin have previously demonstrated that type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into the surrounding connective tissues matrix. The purpose of this study is to determine if individual type VI collagen microfilaments might be connected to banded collagen fibrils, thereby stabilizing the network.

Solid phase binding assays suggest a specific, high affinity interaction between the core protein of the dermatan sulfate proteoglycan decorin and type VI collagen, and immunocytochemical studies in fetal and neonate rabbit cornea suggest an association of decorin with type VI microfilaments. Other studies in skin and perichondrium have localized decorin to a region between the d and e bands of banded collagen fibrils. However, no direct documentation has demonstrated a specific structural interaction between type VI microfilaments and banded collagen fibrils. We, therefore, sought to determine if type VI microfilaments cross banded collagen fibrils between the “d” and “e” bands.

Pre-embedding immunogold-silver (IGSS) techniques are useful to localize antigens in cell monolayers and agarose embedded cell suspensions for transmission electron microscopy. Procedural centrifugations, however, present a challenge when attempting to localize antigens in subcellular fractions. Using a Beckman Airfuge Ultracentrifuge to concentrate the subcellar fraction bands and resuspending the organelles in agarose simplifies IGSS processing and resin embedding procedures.

Control bovine turbinate (BT), and BT cells infected with cytopathic (cp NADL) and non-cytopathic (ncp NY-1) strains of bovine viral diarrhea virus (BVDV) were fractioned according to Bienz et al (1992). Bands containing membrane vesicles (Fig 2) were collected and each fraction band was pelleted at 169,000g for 20 min using an A-95 fixed angle rotor in a Beckman Airfuge Ultracentrifuge. Each fraction pellet was resuspended in 50μl of 30% agarose, solidified, and trimmed to < lmm. IGSS procedures were carried out according to Nanoprobes, Inc., Stoney Brook, NY, and Hearne & Van Campen (1996).

Biotinylation of antibodies to be used in immunolocalization studies is a well established approach to mapping epitopes in biological specimens at both the light and ultrastructural level. Biotinylation of antibodies does not appear to significantly alter their interaction with antigen. A similar approach has been proposed for examining ligand interaction with receptors. Producing a soluble biotinylated ligand that behaves similarly to unlabeled ligand avoids the problems introduced by particulate labels such as ferritin, gold and latex. Particulate labels for ligands can be especially problematic in studies of receptor behavior in live cells since viable cells may directly react to the particulate probe itself. The interaction of plasma fibrinogen with its receptor on blood platelets, GPIIb-IIIa, is required for platelet-platelet cohesion and subsequent formation of a hemostatic platelet plug at sites of vascular injury. To study the events that follow binding of fibrinogen to its receptor5 we have produced a series of fibrinogens biotinylated with molar ratios of biotin:fibrinogen that range from 5 to 50.

Following injury to peripheral nerves, processes involved in regeneration must be activated, restoring the original architecture and synaptic connections of the neuron. This is essential for the efficient operation of the sophisticated communications network of the nervous system. In order to accomplish these tasks, complex changes occur in gene expression. Regenerating neurons shift into a growth mode wherein large amounts of cytoskeletal proteins and other growth-associated proteins are produced. These materials, which are synthesized and produced in the neuronal cell body, are then transferred to the axon via axonal transport systems. Among the cytoskeletal and associated proteins upregulated following injury to the CNS are actin, tubulin and the intermediate filament-associated protein, IFAP-70/280kD. The latter is the subject of this investigation.

Intermediate filaments (IF) are a major constituent of the cytoskeleton of most eukaryotic cells. The IF cytoskeleton is a highly dynamic structure that reorganizes continuously as the cell divides and changes shape in response to its environment.

The standardization of IHC has been a critical issue since 1977. The most significant challenge for the standardization of routine IHC may be the adverse influence of formalin, which is in part due to variable periods of formalin-fixation ranging from 12 hours to days or weeks, resulting in variable intensity of immunostaining for formalin sensitive antigens. The world-wide use of the AR technique for IHC in both clinical and research histopathology has demonstrated that the AR is a breakthrough in pathology. Based on numerous studies of AR-IHC with excellent results for a variety of interesting markers used in diagnostic pathology, the possibility has been raised that AR technique may be useful in the standardization of IHC. The purpose of this study is to demonstrate the possibility of standardization of IHC based on the ‘test battery‘ approach for establishing an optimal protocol of AR in order to achieve a state of ‘maximal retrieval’, showing a equal comparability of AR-IHC staining of archival paraffin-embedded tissues under varying periods of formalin-fixation.

Cisplatin, a potent broad spectrum anti-cancer agent, has been proven effective in the treatments of bladder, lung, ovarian, head and neck, and testicular cancers. Drawbacks of this chemotherapeutic drug are its toxic side-effects, which include severe nausea, vomiting, stomach distention, peptic ulcer and hyperglycemia. Cisplatin treatment induces the hyperglycemia both in clinical situation and the rodents. Because of the role of the inducible nitric oxide synthase (i-NOS) and somatostatin on the dysfunction of the β-cell of the pancreatic islets and the suppression of the insulin secretion, these were studied using immunocytochemical methods before and after cisplatin treatments using rats.

Wistar rats (100-150 g) were divided into two groups of 6 animals each. One group received injections of cisplatin (9 mg/kg) in 0.85% saline in 5 divided dosages over a 5 days period. The other group received the vehicle of injection only. Rats were killed one day after the last injection.