DNA supercoiling factor localizes to puffs on polytene chromosomes in Drosophila melanogaster

DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.     

Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain

Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed throughout all neuronal subpopulations of the CNS but not in non-neuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons.     

Proportionate representation of rDNA and Balbiani ring DNA in polytene chromosomes of Chironomus tentans

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ -free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first-order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta-glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01-0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.     

Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.     

A small influence of HSP90 levels on the trehalose and heat shock element inductions of the yeast heat shock response

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.     

Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34. 8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form "weak points," underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally beta-heterochromatic, acquire a distinct banding pattern, i. e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional alpha-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.     

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts

It is now well recognized that a disorder of left ventricular filling can be sufficient to account for congestive heart failure. Furthermore, evaluation of heart disease would not be complete if it did not include assessment of left ventricular filling, improvement of which probably ensures better control of the heart disease. An efficient and reliable tool for the study of diastolic function is therefore essential. The authors review the current state of knowledge and the more recent developments in Doppler echocardiography in the evaluation of left ventricular diastolic function. After revising the pathophysiology, the methods of studying ventricular filling are described. The recording technique is described, taking into account recent developments in transthoracic and transoesophageal approaches. This investigation provides parameters allowing semiquantitative estimation of filling pressures (mean left atrial pressure, end-diastolic pressure) and reliable evaluation of overall diastolic performance.     

Characteristic expression of 105-kDa heat shock protein (HSP105) in various tissues of nonstressed and heat-stressed rats

BACKGROUND: Transient lower esophageal sphincter relaxations (TLESRs) are the major mechanism permitting gastroesophageal reflux (GER). Little information is available on how anti-reflux surgery affects reflux mechanisms, especially TLESRs. We evaluated the effects of partial fundoplication (Belsey Mark IV) on reflux mechanisms. METHODS: Sixteen patients were prospectively studied before and after Belsey Mark-IV operation by endoscopy, 24-h esophageal pH-metry, and simultaneous recording of pH and lower esophageal sphincter (LES) characteristics by sleeve manometry. RESULTS: The operation was successful in 14 of 16 patients (87%). Fasting and postprandial reflux decreased significantly (P < 0.01) after the operation. Partial fundoplication significantly (P < 0.05) decreased the number of TLESRs per hour in the fasting and postprandial period from 3.2+/-0.4 and 5.6+/-0.5 to 1.7+/-0.3 and 2.8+/-0.4, respectively. The percentage of TLESRs associated with reflux also decreased significantly (P < 0.05). Basal LES pressure increased from 14.7+/-2.1 mmHg to 17.9+/-2.6 mmHg (not significant). CONCLUSIONS: Partial fundoplication controls GER through a reduction in the number of TLESRs and by decreasing the number of relaxations associated with reflux.     

Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines

The heat shock response, which is characterized by the induction of heat shock proteins, is known to affect the ability of tumour cells to cope with potentially adverse conditions such as hypoxia, glucose starvation and cytotoxic immune reactions. To assess the heat shock response of melanoma cells, spontaneous and heat shock induced expression of heat shock proteins was analysed in a panel of 17 human melanoma cell lines. Constitutive expression of HSP27, HSP70, HSC70, HSP90alphabeta and GRP94 proteins was found in all the melanoma cell lines, and HSP70 and HSC70 were also induced by heat shock. The major heat inducible HLA-linked HSP70-1 and HSP70-2 genes were analysed at the mRNA level. Basal expression and inducibility varied between the different melanoma cell lines. In addition, in situ hybridization demonstrated heterogeneous expression of these genes among single cells of a given cell line. In general, each melanoma cell line appears to exhibit an individual type of HSP70 expression that might reflect selection during tumour progression and therapy.     

Expression of the heat shock protein HSP27 in human ovarian cancer

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.     

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.     

Structure variations of short polytene chromosomes in 2 pyrenean populations of Bilobella aurantiaca Caroli (Collemboles)

Structural variations of the short polytene chromosomes VI and VII occur in two Pyrenean populations of Bilobella aurantiaca. They are the result of longitudinal cleavage and also of variations in the number of the thin bands. They are not dependent on ecological factors, but often correlate with the chromosome polytenic stage.     

Molecular characterization of the gene operon of heat shock proteins HSP60 and HSP10 in methicillin-resistant Staphylococcus aureus

Methicillin-resistance of S.aureus (MRSA) was diminished or depressed at 44 degrees C. In order to investigate whether bacterial heat shock response is correlated with methicillin resistance, we examined the inducibility of the heat shock proteins (HSPs) in MRSA, and cloned and sequenced of their genes. A temperature shift from 37 degrees C to 46 degrees C enhanced the production of at least 8 kinds of cytoplasmic proteins as measured with two-dimensional PAGE. The induced protein profile was almost the same as methicillin sensitive S.aureus, and stress conditions due to ethanol, cadmium or low pH. Two of these proteins were HSP60 and HSP10. Their N-terminal amino acid sequences were 79%, and 83%, homologous with thermobacterium PS3, respectively. A positively hybridized 4.2 kbp DNA fragment encoding both proteins was isolated from the chromosomal DNA of MRSA. The resulting sequence revealed two reading frames and showed high homology to those of hsp60 (groEL) and hsp10 (groES) of bacteria (E.coli) and several other species. The genes of HSP60 and HSP10 in S.aureus comprised an operon as in E.coli. The relationship between those HSPs and PBP2' is currently under investigation.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.