In situ hybridization studies of matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1 and type IV collagen in diabetic nephropathy

The effects of UV radiation on humans and animals are receiving increasing attention and much interest has recently been focused on the environmental effects of UV A and UV B. This study compares the in vitro effects of UV A and UV B on the clonogenic survival of two human skin keratinocyte cell lines, HaCaT which are immortal but not tumorigenic and HPV-G transfected keratinocytes which form non malignant tumours in nude mice. The effects were also studied on an EPC fish cell line. The aim of the work was to establish if similar initial and delayed survival responses occurred in both species. The cells were exposed to ultraviolet lamps emitting maximally at 365 nm (UV A) and 302 nm (UV B). Clonogenic survival was determined at appropriate times post exposure. Results for the initial survival curves show that the HaCaT and HPV-G cells did not show any appreciable difference in their response to UV A but the EPC cells were more sensitive at doses < 3000 Jm-2. The EPC cells were more sensitive to UV B at doses < 200 Jm-2 in comparison to the human HaCaT and HPV-G cells with the HPV-G cells showing the most sensitivity to UV B at doses > 200 Jm-2. The possible contribution of lethal mutations (delayed cell death) to the UV radiation response in the HaCaT and EPC cell lines was examined. The results showed that lethal mutations were expressed in the HaCaT cells following exposure to UV A and UV B but no lethal mutations were expressed in the EPC cells.     

Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells

Interleukin-1 (IL-1) is expressed in human endometrium and has been shown to play an integral role in local cellular interactions during implantation. In addition, the matrix metalloproteinase (MMP) and its inhibitor, the tissue inhibitor of metalloproteinase (TIMP), are crucial during implantation, mediating in vitro trophoblast penetration, and are regulated by several cytokines expressed by trophoblast cells. We have investigated the roles of IL-1 beta and transforming growth factor-beta (TGF beta) in regulating TIMP-1, TIMP-3, and 92-kDa type IV collagenase messenger ribonucleic acid (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR. Confluent stromal cell cultures treated with progesterone and estradiol for 9 days were stimulated with IL-1 beta, IL-1 beta plus anti-IL-1 beta antibody, TGF beta, and TGF beta plus anti-TGF beta antibody for an additional 24 h. Competitive complementary DNA fragments were constructed by deletion of a defined fragment from each of the target complementary DNA sequences and coamplified in quantitative competitive PCR as an internal standard. TIMP-1 and TIMP-3, but not 92-kDa type IV collagenase mRNA, were expressed in stromal cells. The 92-kDa type IV collagenase mRNA was only expressed after stimulation with IL-1 beta. IL-1 beta both augmented 92-kDa type IV collagenase mRNA expression and decreased TIMP-1 and TIMP-3 mRNA expression in a dose-dependent manner. Conversely, TGF beta augmented TIMP-1 and TIMP-3 mRNA expression, but did not affect 92-kDa type IV collagenase expression. IL-1 and TGF beta-mediated changes were both neutralized by specific antibodies. These results provide indirect evidence that IL-1 and TGF beta may play crucial roles at the embryo-maternal interface during trophoblast invasion by regulating stromal cell expression of TIMP-1, TIMP-3, and 92-kDa type IV collagenase, all of which are known to be important in trophoblast invasion.     

In situ expression of ICAM-1 and its mRNA in the lung tissue of asthmatic rats

The objective of this work was to determine whether the prevalence of respiratory symptoms differed among workers exposed to different types of metal-working fluids. As part of a mandatory surveillance system for occupational illness, from 1988-1994, the Michigan Department of Public Health received, 86 occupational disease reports of work-related asthma secondary to exposure to metal-working fluids. As part of a public health program, follow-up industrial hygiene inspections, including medical interviews of the workforce, were performed at companies where the reported cases had become ill. Metal-working fluids were the second most common cause of work-related asthma reported in the state. Most of the reports were from the automobile industry. Follow-up inspections were conducted at 37 facilities where the individuals with work-related asthma had worked. Seven hundred and fifty-five workers at these facilities were interviewed. Only one facility was above the allowable oil mist standard. Despite the exposure levels being within the legal limits, approximately 20% of the fellow workers of the reported cases had daily or weekly respiratory symptoms suggestive of work-related asthma. Workers exposed to emulsified, semisynthetic, or synthetic machining coolants were more likely to have chronic bronchitis; to have visited a doctor for shortness of breath; to have visited a doctor for a sinus problem; to be bothered at work by nasal stuffiness, runny nose, or sore throat; and to have an increased prevalence of respiratory symptoms consistent with work-related asthma, compared to workers exposed to mineral oil metal-working fluids. These findings were found in individuals who currently smoked, had never smoked or were ex-cigarette smokers. Further research to determine the chemical components or microbial contaminants responsible for these findings is needed.     

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.     

Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates

The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 (HSP90 alpha) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90 alpha were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90 alpha. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90 alpha and the C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.     

Identification and localization of type IV collagen chains in the inner ear cochlea

The expressions of cysteine dioxygenase (CDO) gene in the liver, lung, skeletal muscle, and kidney were studied by in situ hybridization with a cDNA probe from rat liver CDO under normal conditions. Significant expression of the CDO gene was detected in the liver, lung, and kidney, but not skeletal muscle. In the liver, the signal was confined to the cytoplasm of the hepatocytes. Furthermore, the signal was stronger in the periportal than that in the perivenous areas. In the lung, an intensive signal was found in the bronchiolar epithelium. As to the kidney, an intensive signal was observed in the distal convoluted tubules, while no signal was found in the proximal convultions.     

Cicaprost and the type IV phosphodiesterase inhibitor, rolipram, synergize in suppression of tumor necrosis factor-alpha synthesis

Suppression of tumor necrosis factor-alpha (TNF) synthesis is one major target in pharmacological immunomodulation. We now showed the synergistic suppressive effect of the specific type IV phosphodiesterase inhibitor, rolipram, and of the stable prostacyclin analogue, cicaprost, on TNF synthesis. This effect was seen with lipopolysaccharide and Staphylococcus epidermidis as stimuli in human peripheral blood mononuclear cells and in whole blood. Lipopolysaccharide-induced TNF synthesis by mononuclear cells decreased from 3.4 ng/ml to 1.5 ng/ml in the presence of 100 nM rolipram and to 0.7 ng/ml in the presence of 10 nM cicaprost. The combination of both agents suppressed TNF synthesis more than 10-fold, to 0.3 ng/ml. Synergistic suppression was also demonstrated for TNF mRNA.     

In situ hybridization to detect the mRNA for insulin-like growth factor-I and II in the prostate

In order to well understand the expression of mRNA for insulin-like growth factor I and II in normal and hyperplastic prostate, in situ hybridization were used to detect them in a total of 35 cases of prostate. Both the normal and the hyperplastic prostate expressed the mRNA for IGF-I/II. The mRNA for IGF-I was mainly expressed by the epithelial cells and the IGF-II was mainly expressed by the stromal cells. The hyperplastic tissues expressed higher levels of the mRNA for IGF-I and IGF-II. The results suggested that the IGF might play an important role in the regulation of prostate growth and the pathogenesis of benign prostatic hyperplasia.     

In situ mRNA hybridization technique for analysis of human telomerase RNA in gastric precancerous and cancerous lesions

Telomerase, the ribonucleoprotein enzyme that elongates telomerase, is repressed in normal somatic cells but is reactivated during tumor progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human gastric precancerous and cancerous lesions by using in situ mRNA hybridization (ISH) with avidin-biotin staining. We also examined telomerase activity in these lesions by using hybridization protection assay connected with a telomeric repeat amplification protocol (TRAP/HPA). Analyzed tissue samples were as follows; 132 cases of chronic atrophic gastritis without intestinal metaplasia, 115 incomplete-type intestinal metaplasias, 40 complete-type intestinal metaplasias, 23 hyperplastic polyps, 23 tubular adenomas and 26 adenocarcinomas. In ISH analysis, high levels of hTR expression were observed preferentially in the nuclei at the single-cell level. hTR-expressing cells in carcinomas and adenomas were significantly more frequent than those of the other lesions (P < 0.001). The expression pattern of hTR in carcinoma and adenoma tissues was heterogeneous and similar intratumor heterogeneity was detected in Ki-67 immunoreactivity. Infiltrating lymphocytes in tissue also exhibited high levels of hTR expression. In TRAP/HPA analysis, carcinomas had significantly more frequent positivity for telomerase activity and a higher level of telomerase activity than the other lesions (P < 0.05). However, the amount of telomerase activity did not parallel the expression level of hTR. Our data suggest that hTR expression increases in the early stages of stomach carcinogenesis and that sufficient synthesis of hTR is a prerequisite for telomerase reactivation in tumorigenesis.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Complete sequence of rabbit kidney aminopeptidase N and mRNA localization in rabbit kidney by in situ hybridization

We have isolated and sequenced a full-length cDNA for rabbit kidney aminopeptidase N (APN). The 3-kilobase cDNA contains 12 nucleotides of the 5' noncoding region, a 2898 nucleotide long open reading frame, and 113 nucleotides of the 3' untranslated region. The open reading frame encodes a type II membrane protein of 966 amino acid residues composed of a 10 residue NH2-terminal cytosolic domain, a 23 residue transmembrane domain, and a large 933 residue ectodomain that contains the active site. Rabbit APN has eight potential N-glycosylation sites and seven cysteine residues, one of which is located in the transmembrane domain. Computer analysis showed that the enzymes from human, rat, and rabbit were highly conserved, except for the stalk region immediately downstream from the transmembrane domain. Using in situ hybridization techniques we showed that in rabbit kidney, APN mRNA is present in proximal tubules but not in glomeruli, which corresponds to the localization of the protein observed by immunohistochemistry. Taken together, our results strongly suggest that the expression of APN in kidney is modulated at mRNA levels and not at translational and (or) posttranslational levels.     

Expression and localization of angiotensin II type 1 receptor mRNA in rat ocular tissues

OBJECTIVE: Hereditary hemorrhagic telangiectasia (HHT) is an inherited abnormality passed down as a dominant autosomal feature. Recurrent epistaxis usually constitutes the major clinical manifestation of this disease. The unsatisfactory results of conservative therapy have stimulated a research interest for the role of laser photocoagulation in telangiectatic vessels associated with this clinical entity. METHOD: The Nd:YAG laser was used to treat a group of 11 individuals suffering from HHT, all of whom had been previously treated using other modalities. RESULTS AND CONCLUSION: The excellent results of Nd:YAG laser irradiation are addressed in view of all treatment modalities proposed for the treatment of recurrent epistaxis in HHT.     

In situ localization of WDNM1 and ferritin heavy chain gene expression in mammary gland

In situ hybridization was performed with sections obtained from mammary gland at 10 days of lactation and at 1 and 3 days of involution using either digoxigenin-labeled antisense or sense RNA probe in order to localize expression of WDNM1 and ferritin heavy chain mRNA. The WDNM1 gene was predominantly expressed in the layer of secretory epithelial cells surrounding the lumen of mammary gland alveoli. The lower levels of WDNM1 mRNA were observed at involution day 3 compared to involution day 1. The expression of ferritin heavy chain mRNA also appears to be confined to the epithelial layer of mammary alveoli. The lower levels of ferritin heavy chain mRNA were observed at involution day 3 compared to involution day 1.     

Expression of nm23/NDPK, integrin, type IV collagenase and uPA in transplanted tumors with various metastatic potential in nude mice and surgical specimens

OBJECTIVES: To observe the relationship between the metastasis of human lung carcinoma and the expressions of nm23/NDPK, integrins, type IV collagenase and uPA. METHODS: By ABC immunohistochemical staining, we observed the expressions of nm23/NDPK, integrins, type IV collagenase and uPA in four strains of subcutaneous transplanted tumors and 87 surgical specimens for human lung carcinoma. The metastatic potential of four xenografts varied from low to high among PLA-801C, Anip973, PLA-801D, PLA-801DL. The effects of beta 1 antibody and the RGD peptide on the PLA-801DL xenograft were studied in the nude mice. RESULTS: High expression of nm23/NDPK was observed in the four xenografts and specimens from the 87 patients. No association was found between expression of nm23/NDPK and lymph node metastasis. Low expressions of beta 1 subfamily integrins were observed in the four xenografts. beta 1 subunit antibody and RGD peptide had no effect on metastasis of PLA-801DL xenograft. The expressions of type IV collagenase were positively correlated with their metastatic capacity in the four xenografts and surgical specimens from the 87 patients. Anip973 and PLA-801D showed positive staining to uPA, while PLA-801DL and PLA-801C were negative. CONCLUSIONS: The expression of type IV collagenase is positively correlated with the metastatic capacity of lung carcinoma. Although high expression of nm23/NDPK, low expression of integrins, and various expression of uPA were observed, these were not correlated with metastatic capacity of lung carcinoma.     

In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes

The beta-amyloid (A beta 1-40) peptide has previously been shown to enhance phenylephrine contraction of aortic rings in vitro. We have employed a novel observation, that A beta peptides enhance endothelin-1 (ET-1) contraction, to examine the relationship between vasoactivity and potential amyloidogenicity of A beta peptides, the role played by free radicals and calcium in the vasoactive mechanism, and the requirement of an intact endothelial layer for enhancement of vasoactivity. Rings of rat aortae were constricted with ET-1 before and after addition of amyloid peptide and/or other compounds, and a comparison was made between post- and pre-treatment contractions. In this system, vessel constriction is consistently dramatically enhanced by A beta 1-40, is enhanced less so by A beta 1-42, and is not enhanced by A beta 25-35. The endothelium is not required for A beta vasoactivity, and calcium channel blockers have a greater effect than antioxidants in blocking enhancement of vasoconstriction by A beta peptides. In contrast to A beta-induced cytotoxicity, A beta-induced vasoactivity is immediate, occurs in response to low doses of freshly solubilized peptide, and appears to be inversely related to the amyloidogenic potential of the A beta peptides. We conclude that the mechanism of A beta vasoactivity is distinct from that of A beta cytotoxicity. Although free radicals appear to modulate the vasoactive effects, the lack of requirement for endothelium suggests that loss of the free radical balance (between NO and O2-) may be a secondary influence on A beta enhancement of vasoconstriction. These effects of A beta on isolated vessels, and reported effects of A beta in cells of the vasculature, suggest that A beta-induced disruption of vascular tone may be a factor in the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease. Although the mechanism of enhanced vasoconstriction is unknown, it is reasonable to propose that in vivo contact of A beta peptides with small cerebral vessels may increase their tendency to constrict and oppose their tendency to relax. The subclinical ischemia resulting from this would be expected to up-regulate beta APP production in and around the vasculature with further increase in A beta formation and deposition. The disruptive and degenerative effects of such a cycle would lead to the complete destruction of cerebral vessels and consequently neuronal degeneration in the affected areas.     

Monkey leptin receptor mRNA: sequence, tissue distribution, and mRNA expression in the adipose tissue of normal, hyperinsulinemic, and type 2 diabetic rhesus monkeys

We investigated the discharge morphology and propagation patterns of electroencephalographic seizures of temporal lobe onset in 21 children and young adults who underwent invasive long-term EEG monitoring (LTM). Of those, 15 subsequently underwent anterior temporal lobectomy. The onset was focal in 63%. The most frequent discharge morphology was low amplitude beta (30%) or rhythmic/semirhythmic theta discharge (30%). Thirteen patients displayed several sequences of propagation with different spreading stages along a fixed path. Initial spreading to the ipsilateral frontal lobe was associated with a higher frequency of secondary generalization than initial spreading to the contralateral temporal lobe (P = 0.18). A comparison of 13 patients older than 18 years of age with 8 patients younger than 14 years showed a trend towards a lower rate of propagating from the temporal lobe (P = 0.13) in the younger age group. Discharge morphology was not correlated with age, focality, or outcome of surgery.     

In situ autogenous reconstruction of the thoracoabdominal aorta and branches for treatment of an infected thoracoabdominal aortobifemoral bypass graft

Graft infection is an uncommon but potentially lethal complication of prosthetic aortic repair. We describe a novel technique for upper abdominal aortic and visceral revascularization after percutaneous drainage and antibiotics failed to cure a thoracofemoral prosthetic graft infection. One week after axillofemoral and femorofemoral bypass grafting, the infected thoracoabdominal graft was removed and a bifurcated iliac artery autograft was used to replace the upper abdominal aorta and revascularize the abdominal viscera. The infected graft was removed from the thorax and retroperitoneum, the infection resolved, and the patient remained well until his death of lung cancer 9 years later.