Higher order structure of Balbiani ring premessenger RNP particles depends on certain RNase A sensitive sites.

Specific premessenger ribonucleoprotein (RNP) particles, the Balbiani ring (BR) granules from Chironomus tentans salivary glands, were treated with RNase A to study the effect of RNA strand breaks on the higher order structure of the particles. Isolated, radioactively labeled BR granules, known to sediment at 300 S, were digested with RNase A and centrifuged in sucrose gradients. The fractionated particles were subsequently analyzed using electron microscopy and caesium chloride centrifugation. At a low RNase concentration, most of the 300 S particles disintegrated completely, and no metastable degradation products were observed. At intermediate RNase concentrations, no 300 S particles were left, but a minor fraction of the BR granules had unfolded and sedimented at 160 S. These granules could represent particles modified during the RNase treatment or represent a more slowly degrading subfraction of the particles. At a high RNase concentration, no RNP particles at all remained in the gradient. The rapid disintegration of the majority of the BR granules was investigated further by electrophoretic analysis of RNA in the remaining particles. During the RNase treatment BR granules, still sedimenting at 300 S, accumulated strand breaks; in fact, as many as 50 to 100 nicks in the 37 kb RNA could be tolerated. It was concluded from RNA analyses that the disintegration of the BR granules was not dependent on any single nick in the RNA, nor on the accumulation of a certain number of nicks, but rather on one or a few critical strand breaks. We propose that there are organizing sequences essential for particle integrity; once these sequences are nicked, the premessenger RNP particles are rapidly and completely degraded.

Beta microseminoprotein is not a prostate-specific protein. Its identification in mucous glands and secretions.

Beta microseminoprotein (beta inhibin, PSP94), an unglycosylated protein of 94 amino acids with unknown function, is one of the predominating proteins in the secretion of the human prostate gland. In this work the authors have demonstrated that the expression of beta microseminoprotein is not restricted to the prostate and that the protein has a previously unrecognized widespread occurrence in the human body. According to radioimmunoassay, beta microseminoprotein immunoreactivity is present in many nonprostatic body fluids. The highest concentrations were found in secretions from the respiratory tract; in tracheobronchial fluid sometimes even at concentrations comparable to that in seminal plasma (about 1 g/l). Intermediate concentrations were found in gastric juice and some samples of secretion from the uterine cervix, whereas tears, saliva, pancreatic juice, bile, and mucus from the colon had low concentrations. According to gel chromatography, the molecular size of the beta microseminoprotein immunoreactivity present in tracheal fluid, gastric juice, and secretion from the uterine cervix did not differ from that of beta microseminoprotein in seminal plasma. The beta microseminoprotein immunoreactive component present in gastric juice had the same amino-terminal amino acid sequence as prostatic beta microseminoprotein (14 residues identified in material purified from gastric juice), providing further evidence for chemical identity of a nonprostatic beta microseminoprotein with the prostatic protein. Immunohistochemical staining with affinity-purified antibodies demonstrated the presence of beta microseminoprotein in many tissues, including the goblet cells in the tracheobronchial epithelium, tracheobronchial submucosal glands, certain mucosal cells in the antrum of the stomach, some glands of Brunner in the duodenum, and in parts of the mucosa of the colon. At least in the respiratory tract, the staining was localized to mucus-containing cells. beta microseminoprotein immunoreactivity also was localized to the cilia of the ciliated epithelium in the respiratory tract, the fallopian tubes, and the Gartner ducts of the uterine cervix. The pattern of tissue distribution of beta microseminoprotein found in this work indicates a connection of beta microseminoprotein with mucous secretions.

Ultrastructural localization of the putative precursors of the A4 amyloid protein associated with Alzheimer's disease.

Any explanation of the causes of Alzheimer's disease and of its unique cerebral pathologic features must take into account the distribution and ultrastructural localization of the pre-A4 amyloid proteins in tissues and organs. The authors have analyzed the expression of the pre-A4 amyloid proteins in several tissues by immunogold electron microscopy and by immunofluorescence. For this purpose, they have used a mouse monoclonal antibody and a guinea pig antiserum raised against two synthetic peptides corresponding to two different sequences common to all the full-length forms of the A4 amyloid precursors. They observed a tissue-specific distribution of the secreted and the transmembrane form of the precursors. The authors could determine that the secreted form is generated in vivo within the cytoplasm. In the salivary glands and in the adenohypophysis, all the immunoreactivity is associated with the process of secretion, whereas in the muscle, a staining pattern compatible with the presence of the pre-A4 amyloid proteins in the sarcoplasmic reticulum has been observed. This difference in the localization may reflect tissue-specific processing pathways and suggests that posttranslational modifications such as proteolytic removal of the transmembrane and cytoplasmic domains contribute to the structural and thus functional diversity of the A4 amyloid precursors.

Proteoglycans detected by monoclonal antibodies in adenoid cystic carcinoma of salivary glands.

Thirty-two cases of adenoid cystic carcinoma (ACC) of the salivary glands were examined immunohistochemically by monoclonal antibodies to proteoglycans (PG) such as chondroitin 6 sulfate (C6SPG), chondrotin 4 sulfate (C4SPG), dermatan sulfate (DSPG), heparan sulfate (HSPG) and keratan sulfate (KSPG) in conjunction with specific enzymatic digestion. The cribriform structure of ACC consisted of basaloid, myoepithelium-derived, and luminal tumor cells. The myoepithelial tumor cells were positive for PG, whereas luminal tumor cells were unstained. Occasional pseudocysts also gave positive staining for PG. Tubular structures consisting of modified myoepithelial cells indicated a high intensity reaction for C6SPG and C4SPG, and a slight one for DSPG, HSPG, and KSPG. Immunodepositions in solid and cluster structures were comparatively light in terms of PG. Basement membrane in ACC stained strongly for C4SPG, slightly for C6SPG, and very slightly for DSPG, HSPG, and KSPG. In ACC, immunohistochemical staining of PG was regularly positive in myoepithelium-derived tumor cells, but was irregular in other types of tumor cells.

Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering.

23Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter 23Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial 23Na were observed in the perfused gland at 25 degrees C. The resonance due to intracellular 23Na consisted of two Lorentzian signals stemming from the [1/2 mean value of -1/2[ coherence (sharp resonance) and the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences (broad resonance). The transverse relaxation rate constant corresponding to the [1/2 mean value of -1/2[ coherence was 95 +/- 4 s-1 and that corresponding to the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences was 1360 +/- 75 s-1 (mean +/- S.E., n = 5). The resonance due to the interstitial 32Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N",N",N"'-hexaacetic acid.

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar.

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.

Protein constituents of mosquito saliva: studies on Culex molestus.

The protein constituents of saliva, salivary gland extracts, haemolymph and whole body extract from the Culex molestus mosquito were compared by polyacrylamide gel electrophoresis. When developed by the sensitive silver staining technique, saliva and salivary gland extract were found to contain 15 comparable protein bands. Salivary gland extract contained additional bands, presumed to be structural proteins, and saliva contained some unique protein bands which were not present in the gland extract, possibly originating in the salivary stylet lining. Some differences were found in the protein components of salivary gland extracts prepared from mosquitoes of different ages. Salivary proteins were found to be poorly represented in both haemolymph and whole body extracts. Immunoblotting of salivary gland extract with antibody raised against pure saliva exhibited binding to at least nine protein bands indicating the potential for using salivary gland extracts in place of mosquito saliva for further studies.

Immunohistochemical localization of carcinoembryonic antigen (CEA) in salivary gland tumors.

Carcinoembryonic antigen (CEA) was first isolated from colonic carcinoma and has been used as a diagnostic marker. CEA has also been observed in a variety of epithelial tumors and normal tissues. In this study, CEA was localized by means of immunohistochemical procedures in benign and malignant salivary gland tumors, as well as in normal parotid gland, indicating that CEA is not a reliable marker for differentiation between benign and malignant salivary gland neoplasms.

Bioamines and proteins in the saliva and salivary glands of Palaearctic blackflies (Diptera: Simuliidae).

To elucidate the mechanisms of skin-reactions to blackfly bites, components in the saliva and salivary glands of Simulium lineatum and S. equinum females were studied. Histamine, putrescine, spermine, N1-monoacetyl-spermine, and spermidine were detected in both the saliva and salivary gland extract through high-performance liquid chromatography (HPLC), the content of histamine being much higher than that of the other amines. Whereas histamine concentration was increased by food stimulation and actual feeding, the content of the other amines did not differ from that of non-stimulated flies. S. lineatum and S. equinum females differed significantly in their spermine and spermidine contents, autumn and spring generation females did not differ from each other. The dispersion of histamine in the salivary glands of male and female S. lineatum and S. equinum was demonstrated through histochemical staining. The globular proteins of the salivary glands were fractionated by chromatography and electrophoresis, the molecular weight of proteins and the enzymatic activity of esterase were also determined.

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii.

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.

Surface carbohydrate differences of Glossina salivary glands and infectivity of Trypanosoma brucei gambiense to Glossina.

Incubation of fluorescein- and biotin-lectin conjugates with the salivary glands of Glossina spp has revealed inter- and intraspecific variation in the surface carbohydrates of the glands. The degree of Con A binding to the basal laminae of the glands of the two Glossina palpalis subspecies, G.p. palpalis and G.p. gambiensis was markedly different. The infectivity of T.b. gambiense sensu lato isolates to G.p. palpalis and G.p. gambiensis was compared. G.p. gambiensis from the field and from laboratory colonies transmitted T.b. gambiense sensu lato isolated from patients in Ivory Coast whereas G.p. palpalis appears totally refractory to all T.b. gambiense isolates used. Although the relationship between the surface of the basal laminae of the salivary gland exposed to the haemocoele and trypanosome infection is not known the consistent differences observed in lectin binding to the salivary glands suggest that differences in basic physiology of the glands exist which might correlate with susceptibility to trypanosome infection.

NMR characteristics of intracellular K in the rat salivary gland: a 39K NMR study using double-quantum filtering.

Intracellular K of the perfused rat mandibular salivary gland was measured by 39K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25 degrees C. The resonance due to intracellular K consisted of two Lorentzian signals stemming from the [spin 1/2 to -1/2] coherence (sharp resonance) and the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences (broad resonance). The transverse relaxation time (T2) corresponding to the [spin 1/2 to -1/2] coherence was ca. 2.5 ms, and that corresponding to the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences was ca. 0.4 ms. The relaxation time of the double-quantum coherence of rank 3 (originating from product operators like Ix2Iz) was determined to be ca. 0.2 ms. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 x 10(-8) s and a quadrupolar coupling constant of ca. 1.4 MHz.

Differentiation of rat salivary glands by thermoanalytical analysis.

The water release from the sublingual, parotid and submandibular glands of male and female rats was analyzed by thermal analysis in order to detect the total water content and types. Different types of water, which are increasing from the sublingual to the parotid gland, were found and the relative distribution appeared to be a function of the bond energy of water to glandular components. In addition, evidence of a sexual dimorphism in the rat sublingual gland was demonstrated.

Lack of hemostatic effects of adult and suckling cat salivary gland heteropolysaccharides on blood coagulation.

Like other mammalian salivary glands, the cat submandibular and parotid glands belonging to adult and growing subjects contain heteropolysaccharides with very heterogeneous composition. Such glycoconjugates were this time tested in humans at doses used previously showing anticoagulant properties in rodents and lagomorphs. But surprisingly, no anticoagulant effects could be detected consistently. In fact, neither thromboelastography, nor hemocoagulation screening tests were affected in a statistically significant manner.

Ghilantens: anticoagulant-antimetastatic proteins from the South American leech, Haementeria ghilianii.

Five proteins with anticoagulant and antimetastatic activities were isolated from the salivary glands of the Amazon leech, Haementeria ghilianil. These proteins, designated ghilantens, were co-purified on DEAE-cellulose and heparin-agarose, and were purified by microbore C-18 reverse-phase HPLC. Each variant had a similar molecular weight (18,000), amino acid composition, and a blocked amino terminus. Ghilantens caused a dose-dependent prolongation of the prothrombin time of normal human plasma and blocked the factor Xa-mediated hydrolysis of methoxycarbonyl-D-cyclohexylglycyl-glycl-arginine-p-nitro anillide acetate. Ghilantens were quantitatively absorbed to bovine factor Xa-AffiGel-15 and were eluted with 0.1 mol/L benzamidine, an active-site reversible inhibitor of factor Xa. These findings show that ghilantens can form a reversible association with the enzyme. When administered intravenously to mice by tall vein injection, ghilantens potently suppressed lung metastases of B16-F10 melanoma cells. These findings suggest that ghilantens may have therapeutic value in the treatment of metastatic disease.

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues.

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.

Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins.

Sensory transduction in taste and olfaction, the principal chemical senses, seems to be mediated by membrane-associated proteins on the apical surfaces of the respective receptor cells. The recent isolation and characterization of soluble 'odorant-binding proteins' secreted from the nasal glands of rat, cow and frog, led to the hypothesis that these proteins function as necessary cofactors in olfactory transduction by concentrating and delivering odorants to the receptors. The primary reception of taste stimuli occurs in specialized neuroepithelial receptor cells bundled in taste buds that are clustered in various types of papillae in the lingual epithelium of the tongue. Small tubulo-alveolar salivary glands, the von Ebner's glands, are located beneath the circumvallate and the foliate papillae. Their ducts open exclusively into the trough at the base of the papillae. Taste buds located in the medial and lateral walls of the papillae open with their taste pores into the trough and consequently are in direct contact with the secretions of von Ebner's glands. Here we report the molecular cloning and characterization of a protein of relative molecular mass 18,000 that is highly expressed in von Ebner's glands. Like the odorant-binding proteins, this protein shows similarity to members of a protein superfamily of hydrophobic molecule transporters, indicating that pre-receptor events could also be necessary for the concentration and delivery of sapid molecules in the gustatory system, and emphasizing the close relationship of taste and olfaction.

The rapidly phosphorylated chromosomal 42-kDa protein is a subunit of larger protein complexes.

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster.

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.