Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L.

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

DNA Polymerase4 from the Silk Glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

DNA polymerase alpha-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini.

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequen

An immunoregulatory peptide from salivary glands of the horsefly, Hybomitra atriperoides

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans, and vectors for filariasis. They rely heavily on the pharmacological propriety of their saliva to get blood meat and suppress immune reactions of hosts. Little information is available on horsefly immune suppressants. By high-performance liquid chromatography (HPLC) purification coupling with pharmacological testing, an immunoregulatory peptide named immunoregulin HA has been identified and characterized from salivary glands of the horsefly of Hybomitra atriperoides (Diptera, Tabanidae). Immunoregulin HA could inhibit the secretion of interferon-gamma (IFN-gamma) and monocyte chemoattractant protein (MCP-1) and increase the secretion of interteukin-10 (IL-10) induced by lipopolysaccharide (LIPS) in rat splenocytes. IL-10 is a suppressor cytokine of T-cell proliferative and cytokine responses. IL-10 can inhibit the elaboration of pro-inflammatory cytokines. Immunoregulin HA possibly unregulated the IL-10 production to inhibit IFN-gamma and MCP-1 secretion in the current experiments. This immunosuppression may facilitate the blood feeding of this horsefly. The current works will facilitate to understand the molecular mechanisms of the ectoparasite-host relationship. 2008 Elsevier Ltd. All rights reserved.

Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-gamma production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands

Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their sal

Two Immunoregulatory Peptides with Antioxidant Activity from Tick Salivary Glands

Ticks are blood-feeding arthropods that may secrete immunosuppressant molecules, which inhibit host inflammatory and immune responses and provide survival advantages to pathogens at tick bleeding sites in hosts. In the current work, two families of immunoregulatory peptides, hyalomin-A and -B, were first identified from salivary glands of hard tick Hyalomma asiaticum asiaticum. Three copies of hyalomin-A are encoded by an identical gene and released from the same protein precursor. Both hyalomin-A and -B can exert significant anti-inflammatory functions, either by directly inhibiting host secretion of inflammatory factors such as tumor necrosis factor-alpha, monocyte chemotectic protein-1, and interferon-gamma or by indirectly increasing the secretion of immunosuppressant cytokine of interleukin-10. Hyalomin-A and -B were both found to potently scavenge free radical in vitro in a rapid manner and inhibited adjuvant-induced inflammation in mouse models in vivo. The JNK/SAPK subgroup of the MAPK signaling pathway was involved in such immunoregulatory functions of hyalomin-A and -B. These results showed that immunoregulatory peptides of tick salivary glands suppress host inflammatory response by modulating cytokine secretion and detoxifying reactive oxygen species.

Mitochondrial mutations in adenoid cystic carcinoma of the salivary glands.

BACKGROUND: The MitoChip v2.0 resequencing array is an array-based technique allowing for accurate and complete sequencing of the mitochondrial genome. No studies have investigated mitochondrial mutation in salivary gland adenoid cystic carcinomas. METHODOLOGY: The entire mitochondrial genome of 22 salivary gland adenoid cystic carcinomas (ACC) of salivary glands and matched leukocyte DNA was sequenced to determine the frequency and distribution of mitochondrial mutations in ACC tumors. PRINCIPAL FINDINGS: Seventeen of 22 ACCs (77%) carried mitochondrial mutations, ranging in number from 1 to 37 mutations. A disproportionate number of mutations occurred in the D-loop. Twelve of 17 tumors (70.6%) carried mutations resulting in amino acid changes of translated proteins. Nine of 17 tumors (52.9%) with a mutation carried an amino acid changing mutation in the nicotinamide adenine dinucleotide dehydrogenase (NADH) complex. CONCLUSIONS/SIGNIFICANCE: Mitochondrial mutation is frequent in salivary ACCs. The high incidence of amino acid changing mutations implicates alterations in aerobic respiration in ACC carcinogenesis. D-loop mutations are of unclear significance, but may be associated with alterations in transcription or replication.