Generation and subcellular distribution of histamine in human blood monocytes and monocyte subsets

OBJECTIVE AND DESIGN: This study was designed to establish the sites of formation and storage of histamine and histidine decarboxylase (HDC) in human monocytes and two of their subsets. MATERIALS AND METHODS: The experiments were carried out using monocytes from buffy coats of healthy blood donors. Histamine was quantitated by RIA, HDC activity by the formation of histamine. RESULTS: The monocyte subtype RM3/1 contained significantly more histamine than the subset 27E10 (0.041+/-0.025 vs. 0.005+/-0.004 pg/cell, p < 0.05) and also more HDC activity and HDC mRNA. After fractionation of monocyte homogenates in a discontinuous Percoll gradient or by differential centrifugation more than 80% of both, HDC activity and histamine, were recovered from the cytosolic fractions. About 50% of this histamine was found to be bound to proteins. CONCLUSIONS: In monocytes histamine and HDC are colocalized in the cytoplasm indicating a subcellular distribution different from mast cells or basophils. The data also show that histamine is synthesized by the monocytes themselves.     

Phosphatidylinositol transfer protein in lung: cellular and subcellular localization

Determination of the cellular distribution of phosphatidylinositol transfer protein in rat lung by immunocytochemistry revealed that the protein is more readily observed in the nonciliated bronchial epithelial cells (Clara cells) than in other lung cells. By light microscopy, the phosphatidylinositol transfer protein (PtdIns-TP) was localized to the dome-shaped apical region of Clara cells that were identified by staining with an antibody to Clara cell protein. Further investigation by electron microscopy revealed that the PtdIns-TP accumulated at the limiting membrane surrounding secretory granules and at the apical plasma membrane. This localization is compatible with the proposed roles for PtdIns-TP in formation of vesicles and exocytosis of secretory granules and, when considered in the context of the proposed role of PtdIns-TP in phosphatidylinositide metabolism, suggests that phosphatidylinositides may be involved in the mechanisms regulating Clara cell secretion.     

Platelet multielemental composition, lability, and subcellular localization

Diagnostic X-ray spectrometry (DXS), based on X-ray fluorescence, was used to quantitate directly the multiple elemental composition of washed, intact human platelets (n = 16), with the following results: K = 3.08 +/- 1.00 mg/g, Ca = 1.18 +/- 0.29 mg/g, Zn = 35 +/- 9 micrograms/g. These values show that washed platelets contain significant pools of K, Ca, and Zn, the latter some 30-60-fold higher than plasma levels. Dialysis of whole platelets against cation exchange resin (Chelex-100) did not extract Ca(II) and Zn(II) sequestered within whole cells. To identify the subcellular locale of the elements, platelet lysate was subjected to 30-70% sucrose gradient ultracentrifugation and subcellular enriched fractions were obtained. Fractions were analyzed by DXS (for elements), electron microscopy (for dense granules), and subcellular markers fibrinogen and von Willebrand factor. In contrast to Ca and K, which accumulate in the dense granules and the cytoplasm, respectively, Zn appears to be distributed in the alpha-granules (40%) and the cytoplasm (60%). The subcellular distribution of Zn(II) is discussed within the context of the sensitivity of platelet response to the availability of Zn(II) and the platelet release reactions following stimulation.     

Cyclins: relevance of subcellular localization in cell cycle control

OBJECTIVE: To develop a filter utilizing mathematical theory to extract the skeletal patterns of trabecular bone. METHODS: Studies of morphology in the extraction of patterns of calcification in mammograms provided the theoretical framework. Using these studies as a basis, a morphological filter was applied to extract skeletal patterns from digital images of trabecular bone. Sequential images (subset) were combined in a structured fashion to create an aggregate (sumset) which compared with the original images, skeleton and line skeleton images. RESULTS: Binary images of the skeletal patterns in continuous, round and mesh-like forms were obtained from the original images by processing with the skeleton operation using a disc-shaped single structuring element. The line skeleton operation using line structuring elements with constant directions allowed the extraction of linear and discontinuous patterns. Both the skeleton and line skeleton operations extracted binary subset images which depicted skeletal patterns correlating with the operation sequence. CONCLUSIONS: Modification of the morphological filter enhanced the extraction of skeletal characteristics of trabecular bone. A morphological filter may be a useful adjunct in computer-aided structural analysis of bone.     

c-Cbl tyrosine phosphorylation and subcellular localization in human primary leukemic cells

Several studies indicate that a number of signal-transducing molecules involved in the proliferation, differentiation, and functional activation of normal hemopoietic cells may be constitutively activated in primary leukemic cells and play a role in the outcome or in the progression of these neoplastic disorders. In this study we show that the product of the proto-oncogene c-Cbl, whose function is still unknown, is constitutively tyrosine phosphorylated not only in cells from chronic myelogenous leukemias (CMLs) in the blast phase, but also in cells from acute myeloblastic leukemias (AMLs), Ph-negative acute T-lymphoblastic leukemias (T-ALLs), and Ph-negative pre-B lymphoblastic leukemias (pre-B ALL). Moreover, in acute leukemia cells, c-Cbl was not stably complexed with the tyrosine-phosphorylated adaptor protein CrkL. The analysis of Grb2/c-Cbl interaction demonstrated that, in both acute leukemia and CML blasts, c-Cbl was stably complexed with the N-terminal Src homology (SH) 3 domain of Grb2 and, in blasts from ALL patients, with the Grb2 SH2 domain. The analysis of c-Cbl subcellular distribution showed that in all cases of leukemia tested, as well as in growth factor-stimulated M-07e cells, c-Cbl was present in the cytosolic, in the membrane, and in the detergent-insoluble fractions. Finally, in polymorphonuclear neutrophils (PMNs) from CML patients, c-Cbl was found stably associated with the detergent-insoluble fraction, whereas in PMNs from normal donors, it was detected only in the cytosolic fraction. Our findings that c-Cbl is constitutively tyrosine phosphorylated and associated with the detergent-insoluble fraction in AML and ALL blasts and in PMNs from CML patients suggest that this event represents a common step in the neoplastic transformation of both myeloid and lymphoid progenitor cells.     

Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.     

Distribution and subcellular localization of a lysosome-associated protein in human genital organs

The management of breast cancer is becoming increasingly complex, almost year by year. Previous paradigms have held that issues were dichotomous (e.g., good versus bad). These are being challenged constantly by more complex models that demand more than a duality of choice in therapeutic decision making. Most importantly, the determinants of local failure after conservative treatment are quite different from the determinants of survival. While we develop enhanced chemotherapy for immediate life-threatening disease, we also accepting subtypes of breast cancer that are of little threat to life and need little treatment. With the establishment of the efficacy of systemic chemotherapy, the question of whether some patients may be helped with chemotherapy becomes extremely important. The list of possible clinically useful subcategories is growing and is under active development. However, the prognostic factors in use have been validated repeatedly. The two major elements of anatomic staging--size and lymph node status--interact powerfully with histologic categories of grade and special type. In the area of small tumors, these associations indicate lesions that have almost no likelihood of association with distant failure, at least within 5 years.     

Biphasic subcellular localization of the DAZL-related protein boule in Drosophila spermatogenesis

The Drosophila boule gene is expressed exclusively in the male germline and encodes an RNA binding protein closely related to the mammalian fertility factors encoded by the DAZ (Deleted in Azoospermia) and DAZL (DAZ-like) genes. Mutation of boule blocks both meiotic divisions. Differentiation nonetheless continues, resulting in tetraploid spermatids that fail to mature into sperm. We have found that Boule localizes premeiotically to a perinucleolar region and then translocates to the cytoplasm at the onset of meiosis. We show that deletion of the Y chromosome ks-1 fertility locus eliminates Boule nuclear localization, although it does not perturb entry into meiosis. Based on these observations we propose that Boule acts in the cytoplasm to regulate the stability or translation of messenger RNA encoding an essential meiotic factor.     

Cloning, subcellular localization and functional expression of human RNase HII

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.     

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.     

Characterization and subcellular localization of ribonuclease H activities from Xenopus laevis oocytes

Ribonuclease H activities present in fully grown Xenopus oocytes were investigated by using either liquid assays or renaturation gel assays. Whereas the test in solution detected an apparently unique class I ribonuclease H activity, the activity gels did not detect this enzyme but another one with the molecular weight expected for a class II ribonuclease H. The ribonuclease HI was found to be primarily concentrated in the germinal vesicle, but around 5% of this activity was detectged in the cytoplasm and may correspond to the activity involved in antisense oligonucleotide-mediated destruction of messenger RNAs. The concentration of this class I ribonuclease H in oocytes is similar to that in somatic cells. The class II ribonuclease H remained undetectable by the test in solution because its activity was cryptic. On activity gel, a polypeptide with the apparent molecular mass of 32 kDa, expected for a ribonuclease HII, was found to be concentrated in mitochondria although no RNase H activity could be detected by using the liquid assay. Based on sedimentation studies, we hypothesize that the apparent absence of RNase H activity in solution could be the result of the association of this 32-kDa polypeptide with other polypeptides, or possibly nucleic acids, to form a multimer of, until now, unknown function.     

D1 receptor in interneurons of macaque prefrontal cortex: distribution and subcellular localization

Working memory performance is influenced by dopamine activation of D1 family dopamine receptors in the prefrontal cortex; working memory performance is maximal at moderate stimulation of D1 family receptors and is reduced by either higher or lower levels of D1 stimulation. The neuronal mechanisms that underlie this complex relationship are not yet understood. Previous work from this laboratory has demonstrated that the D1 family receptors, D1 and D5, are located in different compartments of pyramidal cells. Here we use an antibody specific to the D1 receptor and double-label immunohistochemistry at the light and electron microscopic level to demonstrate that D1-like immunoreactivity (D1-LIR) is also present in interneurons. D1 receptor is prevalent in parvalbumin-containing interneurons and is less common in calretinin-containing interneurons. At the ultrastructural level, D1-LIR is found associated with the Golgi apparatus and endoplasmic reticulum in the soma, with the membranes of vesicles in proximal dendrites, and with the plasma membrane on distal dendrites, where it is often located near asymmetric synapses. In addition, D1-LIR is also seen in presynaptic axon terminals, which give rise to symmetric synapses onto dendritic shafts and soma. These results raise the possibility that the circuit basis of working memory in the prefrontal cortex involves a D1-mediated inhibitory component.     

Differential subcellular p53 localization and function in N- and S-type neuroblastoma cell lines

Neuroblastoma (NB) cells in vitro are capable of bidirectional transdifferentiation, resulting in two distinct, yet reversible, phenotypes of neuroblastic (N-type) and nonneuronal (S-type) Schwann-like cells. Our previous studies suggested that the wild-type p53 protein is subject to differential regulation in a subset of neuronal cell types. To further test this hypothesis, we compared p53 function in three matched pairs of N- and S-type cell lines, each pair originating from an individual NB tumor. Our data show that although p53 remains cytoplasmically sequestered in a punctate pattern in N-type cells after DNA damage, the protein is diffusely distributed in the S-type cells and is additionally capable of translocating to the nucleus and mediating a biological response to this damage. Our data, therefore, suggest that the p53 protein may be differentially regulated by a neuronal cellular environment and that the sequestration of p53 in NB may be reversible.     

Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis

A genetic etiology in autism is now strongly supported by family and twin studies. A 3:1 ratio of affected males to females suggests the involvement of at least one X-linked locus in the disease. Several reports have indicated an association of the fragile X chromosomal anomaly at Xq27.3 (FRAXA) with autism, whereas others have not supported this finding. We have so far collected blood from 105 simplex and 18 multiplex families and have assessed 141 patients by using the Autism Diagnostic Interview-Revised (ADI-R), the Autism Diagnostic Observation Scale, and psychometric tests. All four ADI-R algorithm criteria were met by 131 patients (93%), whereas 10 patients (7%) showed a broader phenotype of autism. Southern blot analysis was performed with three different enzymes, and filters were hybridized to an FMR-1-specific probe to detect amplification of the CCG repeat at FRAXA, to the complete FMR-1 cDNA probe, and to additional probes from the neighborhood of the gene. No significant changes were found in 139 patients (99%) from 122 families, other than the normal variations in the population. In the case of one multiplex family with three children showing no dysmorphic features of the fragile X syndrome (one male meeting 3 out of 4 ADI-algorithm criteria, one normal male with slight learning disability but negative ADI-R testing, and one fully autistic female), the FRAXA full-mutation-specific CCG-repeat expansion in the genotype was not correlated with the autism phenotype. Further analysis revealed a mosaic pattern of methylation at the FMR-1 gene locus in the two sons of the family, indicating at least a partly functional gene. Therefore, we conclude that the association of autism with fragile X at Xq27.3 is non-existent and exclude this location as a candidate gene region for autism.     

Correlation of subcellular and intratumoral photosensitizer localization with ultrastructural features after photodynamic therapy

Photodynamic therapy (PDT) of cancer typically involves systemic administration of tumor-localizing photosensitizers followed 48-72 h later by exposure to light of appropriate wavelengths. Knowledge about the distribution of photosensitizers in tissues is still fragmentary. In particular, little is known as to the detailed localization patterns of photosensitizers in neoplastic and normal tissues as well as the relationship between such patterns and the actual targets for the photosensitizing effect. This review focuses on ultrastructural features seen in treated cells and tumors. An attempt is made to correlate these findings with the subcellular/intratumoral localization pattern of the photosensitizers in tumor cell lines in vitro and in tumor models in vivo. Several subcellular sites are main targets of PDT with different sulfonated aluminum phthalocyanines (AIPcSn) in the human tumor cell line LOX. Nuclei are not among the primary targets. Overall, the ultrastructural changes correlate well with the data about the subcellular localization patterns for each analogue of AIPcSn in the same cell line. Similar findings are also obtained for the family of sulfonated mesotetraphenylporphines (TPPSn) in the NHIK 3025 cell line. The mechanisms involved in the killing of tumors by PDT seem to be a complex interplay between direct and indirect (via vascular damage) effects on neoplastic cells according to the intratumoral localization pattern of the applied dye. Several factors can affect the localization pattern of a drug, such as its chemical character, the mode of drug delivery, the time interval between drug administration and light exposure, and tumor type. Furthermore, whether local immune reactions (such as macrophages) and apoptosis (programmed cell death) are involved in the destruction of neoplastic cells by PDT in vivo is still an enigma. A general model for PDT-induced tumor destruction is suggested.     

Differential subcellular localization of the survival motor neuron protein in spinal cord and skeletal muscle

OBJECTIVE: To determine the outcome, safety, and possible cost savings of patients undergoing weekend or holiday exercise treadmill testing. DESIGN: Medical records of all 195 patients scheduled for weekend and holiday exercise testing were reviewed, and 77.9% of patients were contacted by telephone to ascertain medical outcomes and need for further emergency department or inpatient care. Costs were calculated from estimates of days of hospitalization saved and incremental costs incurred in conjunction with weekend or holiday testing. SETTING: Urban tertiary care academic medical center. PATIENTS: A total of 195 patients were scheduled for testing, and 181 tests were performed. Over three quarters (75.1%) of patients underwent testing for assessment of chest pain. Other indications included risk stratification after myocardial infarction or coronary angioplasty or prior to noncardiac surgery, or evaluation for arrhythmias, dyspnea, or syncope. MEASUREMENTS AND MAIN RESULTS: Outcomes included results and complications of testing, hospital course after testing, subsequent emergency department visits and readmissions, myocardial infarction, need for cardiac catheterization or revascularization, and mortality. No complications were noted during testing. In 136 patients tested for the indication of chest pain, 90 (66.2%) had negative tests, 39 (28. 7%) were intermediate, and 6 (4.4%) were positive for ischemia. Same day discharge occurred in 115 (84.6%) of the patients, saving an estimated 185 days of hospitalization ($316.83 per patient tested). Event rates over the 6 months following discharge were low. CONCLUSIONS: Weekend and holiday exercise testing is a safe and effective means of risk stratification prior to hospital discharge for patients with chest pain. It also reduces length of stay and is cost saving.     

Primary CNS lymphoma. Infratentorial localization and prolonged survival

A case of a 35-year-old woman presenting infratentorial CNS lymphoma is reported. In 1990 she complained of diplopia, blurred vision and left horizontal nistagmus. An MRI disclosed a lesion in the medulla, pons, and cerebellar vermis and peduncles. Although no treatment was administered, a later RMI showed less extension of the tumor. One year after clinical diagnosis, she received corticosteroids; during the second year a stereotaxic biopsy of the cerebellar lesion was done showing a diffuse B cell non-Hodgkin's lymphoma. A whole brain irradiation was given (50 Gy). She did well for five years, and remains alive (79 months).