Adult Human Organs Research Articles

Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in late pathological stage human prostate cancer.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme best known to many investigators as a 'housekeeping' gene used as a loading control on northern blots. Prior studies, however, have shown that GAPDH RNA levels vary greatly among different prostate cancer cell lines. We undertook this study to determine the level of GAPDH gene expression within primary human prostate tumors and to determine the validity of using GAPDH as a loading control for northern analysis of human prostate cancer specimens and normal human organs. We found that GAPDH expression was significantly higher in specimens from patients with pathological stage C and D prostate cancer than those with stage B disease. Within the human prostate cancer cell lines TSUPr1, DU145, LNCaP and PC-3 and a normal neonatal prostate epithelial cell line FNC 267beta1, LNCaP cells expressed the highest level of GAPDH but all cell lines, including the normal neonate prostate cell line had very strong GAPDH gene expression. GAPDH RNA levels were highly variable among 16 normal human adult organs and four human fetal organs examined. We conclude that GAPDH RNA levels in human prostate tumors correlate with pathologic stage and that GAPDH should not be used as a loading control in northern blot experiments. Other controls, such as beta-actin or 28s ribosomal RNA, would be more suitable for such purposes.

Differential patterns ofHOX gene expression are associated with specific integrin and ICAM profiles in clonal populations isolated from a single human melanoma metastasis

Homeobox-containing genes comprise a gene family coding for transcription factors involved in normal development. Class I human homeobox (HOX) genes display a peculiar chromosomal organization, perhaps directly related to their function. Aberrant expression of homeobox genes has been associated with both morphological abnormalities and oncogenesis. We have reported that HOX gene expression is (i) specific for normal adult human organs (kidney, colon, lung) and (ii) altered in cancer specimens according to their histological type and stage of tumor progression. Here, we have investigated whether patterns of HOX gene expression are associated with tumor heterogeneity by analyzing the expression of the entire panel of 38 HOX genes in clones isolated from a single human metastatic melanoma call line (Me 665/2). The differential expression of a block of genes located at the 5' end of the HOX C locus allows melanoma clones to be classified into 2 major groups. The 2 patterns of HOX gene expression are inversely associated with 2 distinct surface phenotypes for integrins (VLA-2, VLA-5 and VLA-6) and the adhesion molecule ICAM-1. The genes of the HOX C locus are silent in the clones with high levels of integrins VLA-2, VLA-5 and VLA-6 and of the adhesion molecule ICAM-1 but actively expressed in the clones with low levels of ICAM-1 and lacking VLA-2, VLA-5 and VLA-6. Our results indicate that HOX gene expression reflects the intra-tumor heterogeneity of melanoma clones and suggest that the expression of surface molecules involved in cell-cell and cell-matrix interactions may be related to the patterns of HOX gene expression.

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183).

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.

Microtubule-associated proteins in adult human sensory organs.

The distribution of microtubule-associated proteins MAP-1 and MAP-2 was analysed with immunomorphological techniques in the serially sectioned adult human membranous labyrinth. In the organ of Corti, monoclonal antibodies to MAP-1 did not stain. Positivity for MAP-2 occurred in the entire outer hair cell cytoplasm, in the inner hair cells (?), in the nerve fibres and in the cytoplasm of epithelial cells of the spiral prominence. In addition, staining for MAP-2 was identified in many (but not all) cells or Reissner's membrane. Immunofluorescence for MAP-1 occurred in the supporting cells of the cristae and maculae interpreted to be localized in the apical region adjacent to the sensory cells. Thus, the distribution of MAP-1 and MAP-2 in the adult human membranous labyrinth was the same as described for several animal species with regard to the cochlea. In contrast to such a pattern, both MAP-1 and MAP-2 were identified in the human vestibular organs, thus identifying a subpopulation of centrally located nerve calyces and possibly also the apical portion of vestibular hair cells.

Calbindin-like immunoreactivity in epithelial cells of the newborn and adult human vomeronasal organ

The vomeronasal organs (VNOs) of two humans, a male neonate and a female adult, were examined for immunolocalization of calbindin-D28k (calbindin) which has been immunolocalized to VNO receptor cells in other mammals. The present study demonstrates that epithelial cells within the VNOs of both subjects expressed calbindin-like immunoreactivity. These results suggest that human VNO epithelial cells of both genders express calbindin during development and in the adult.

Tissue-differential expression of two distinct genes for human IMP dehydrogenase (E.C.1.1.1.205)

Human IMP dehydrogenase (E.C.1.1.205) is recently regarded as a potent targeting enzyme for immunosuppressive drugs. Tissue differential expressions of human type I and type II IMP dehydrogenase were investigated in sixteen human adult organs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes) and five human fetal organs (heart, brain, lung, liver, kidney) using Northern blot analysis. In all tissues examined in this study, the sizes of mRNAs of each isoform were identical, respectively. The 2.3 kb type II mRNA was shown predominantly, and the 3.5 kb type I mRNA level was lower than type II in most human tissues examined. In contrast, type I IMPDH gene expressed higher than type II in peripheral blood leukocytes, uniquely. We also demonstrated that both type I and type II IMPDH genes are widely distributed among various species by Southern blot analysis. Interestingly, type I IMPDH gene may have multiple gene families in primates.

Peripheral human CD5+ and CD5- B cells may express somatically mutated VH5- and VH6-encoded IgM receptors.

Previous studies have indicated that a sizable fraction of adult human peripheral blood B cells may express IgM receptors encoded by somatically mutated V regions. From these studies it was uniquely associated with the peripheral blood B cell compartment, was uniquely associated with the peripheral blood B cell compartment, associated with particular VH gene segments and/or B cell subpopulations. We have addressed these issues by analyzing > 80 VH5 and VH6-encoded mu transcripts from unseparated peripheral blood, tonsil, and spleen B cells, as well as from B cells separated on the basis of CD5 Ag expression. The results demonstrate that somatically mutated VH5 and VH6 regions are ubiquitously expressed in IgM-bearing B cells in all peripheral adult human lymphoid organs, and that the occurrence of somatic mutations does not segregate with either CD5+ or CD5- B cell populations. The distribution and nature of mutations, as well as the occurrence of clonally related but divergent transcripts suggests that at least some of the mutations were selected by Ag.

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes.

Plasma membranes (1-2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 microM [alpha-32P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37 degrees C for 1 h; during this incubation, the [32P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [32P] guanosine diphosphate (GDP). [32P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca2+, Mg2+, Mn2+, and Zn2+ maximally inhibited GDP release by 80-90%, whereas, 5 mM Cu2+ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One microM Gpp(NH)p (5'-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [32P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1-100 nM glucagon (used as a positive control) stimulated [32P]GDP release by about 17% (P < .05); similarly, 0.1-100 nM insulin stimulated [32P]GDP release by 10-13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [32P]GDP release by 7-10%. In the rat membranes, 10 nM insulin stimulated [32P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [32P]GDP release by about 9% at 2 and 4 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of the human vomeronasal organ

Virtually all vertebrates have a vomeronasal system whose involvement in pheromone detection plays a crucial role in reproduction. In humans, the vomeronasal organ has been assumed to be vestigial or absent and without functional significance. In the present study involving over 400 subjects, vomeronasal pits were observed in all individuals except those with pathological conditions affecting the septum. Electron microscopy of the adult human vomeronasal organ indicates the presence of two potential receptor elements in the pseudostratified epithelial lining: microvillar cells, and unmyelinated, intraepithelial axons. In addition, unmyelinated axons are common in the lamina propria surrounding the organ. They appear to constitute the components essential for a functional chemosensory system, and may thus provide the basis for a pheromone detection system as in other animals.

Progress in Temporal Bone Histopathology

A new tissue processing technique is described for immunohistochemistry of the adult human inner ear, avoiding the immuno-compromising step of prolonged decalcification. the procedure allows reliable investigation of frozen sections of the chemically fixed, non-decalcified, adult human cochlea and vestibular sense organs. Preservation of immu-noreactivity, combined with satisfying tissue morphology, is illustrated by the immunohistochemical localization of the intermediate filament constituents cytokeratins and neurofilament proteins throughout the membranous labyrinth.

Das Zytokeratinskelett des menschlichen Corti-Organs und seine funktionelle Bedeutung*,**

In the adult human organ of Corti cytokeratin (CK) is expressed by all supporting cells enclosing it like a shell. The pattern of immunoreactivity clearly demonstrates a quantitative gradient in the expression of CK, with more CK at the apex than at the base of the cochlea. Predominantly in the apical cochlear turns, the CK-shell separates the compartment of the inner hair cells from that of the outer hair cells. Ultrastructurally, the supporting cells contain a loose fibrillary network which apically is oriented toward the desmosome chain and which can be clearly distinguished from the well-known tubular filaments (microtubuli). Some supporting cells show centrioles. Both the expression of CK and the presence of centrioles indicate a possible potential for cell regeneration. Ultrastructurally, outer hair cells and Deiters' cells show features of a specialized contact zone which might be of functional significance regarding the contractile abilities of the outer hair cells. In the human organ of Corti, vimentin is expressed only by the inner and outer pillar cells. These cells thus express CK together with vimentin. The distinct shell configuration of the CK network in the organ of Corti gives it a tonotopically related difference in rigidity which not only must be of importance for cochlear perception of sound but also could explain the reduced vulnerability of the ear to low frequencies.

Studies of the isolation and viability of human islets of Langerhans.

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Immunohistochemical Characterization of a 183 KD Myeloid-Specific–DNA-Binding Protein in B5 Fixed, Paraffin-Embedded Tissues, and Bone Marrow Aspirates by Monoclonal Antibody BM-1

AbstractA monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-0 showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Histochemical detection of alpha-D-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-alpha-D-glucoside.

5-Br-4-Cl-3-Indoxyl-alpha-D-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fuchsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid alpha-D-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane alpha-D-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane alpha-D-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid alpha-D-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal alpha-D-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.

Immunohistochemical identification of tissue culture cells.

SummaryCell lines of different species have been shown to be differentiated by immunohistochemical technics using properly absorbed antisera. In the work reported here the antisera were prepared against human and mouse tissues and reacted strongly with the connective tissue of the corresponding species in sections of adult human or adult mouse organs, respectively. These antisera also reacted individually with tissue culture cells of human and mouse organs. In all cases the use of the proper tissue in sufficient amounts to absorb the antiserums plays a crucial part in being able to demonstrate the specific staining of cells. Differentiation can be made even within the cell lines of a single species when unique components are present in one of them. The use of anti-myosin serum to detect muscle cells is an example. The technic of using paired labeled antiserum quickly and effectively demonstrates that very slight contamination can be recognized. Such a small contamination would be difficult to detect by ot...