Cytodifferentiation in the accessory glands of Tenebrio molitor: VIII. Crossed immunoelectrophoretic analysis of terminal differentiation in the postecdysial tubular accessory glands

The terminal differentiation of the tubular accessory glands in the postecdysial male mealworm beetle, Tenebrio molitor, is characterized by the synthesis of four major groups of proteins as analyzed by one-dimensional SDS-poly-acrylamide gel electrophoresis. On two-dimensional gels (SDS and pI), two of these groups of proteins (A and B) are acidic in nature and have apparent MWs of 17,500 and 18,800, respectively. A third protein group (C) is heterogeneous with respect to isoelectric point (neutral to basic) and has a MW of about 21,900. The fourth class of proteins (D) is divisible into two subgroups on the basis of isoelectric points and has MWs ranging from 26,400 to 29,100. Antibodies have been produced to the soluble portion of mature tubular accessory glands. The preparations of heterologous antisera have been shown to be gland specific following absorption with bean-shaped accessory gland homogenates and ammonium sulfate fractionation. The first appearance and subsequent accumulation during postecdysial maturation of some of these proteins have been determined by crossed immunoelectrophoresis. The A and B proteins are detected immunologically, initially in the late pupa, and their quantities remain unchanged until shortly after adult eclosion. During the 8 days following adult eclosion each of these proteins increases in quantity from ～10 to ～7000 ng. A third immunoreactive protein appears in the 2-day old adult and increases to ～960 ng over the following 6 days. The identity of this antigen is uncertain although it may represent either the C or the D protein. Based on crossed immunoelectrophoresis, it is evident that at least two of these tubular accessory gland antigens appear to contribute to the spermatophore.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.     

Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds

There is much interest in developing methods amenable to amplifying nucleic acids by the polymerase chain reaction (PCR) in small volumes in microfabricated devices. The use of infrared-mediated temperature control to accurately thermocycle microliter volumes in microchips fabricated from polyimide is demonstrated. Amplification of a 500-base-pair fragment of lambda-phage DNA was achieved in a 1.7-microl chamber containing a thermocouple that allowed for accurate control of temperature. While previous work showed that Taq polymerase was inactivated when in direct contact with the thermocouple, this was circumvented with the polyimide chip by the addition of polyethylene glycol as a buffer additive. This, consequently, allowed for adequate amounts of PCR product to be observed after only 15 cycles, with a total time for amplification of 240 s.     

Pterospora floridiensis,a new species of acephaline gregarine (Apicomplexa) from the maldanid polychaete Axiothella mucosa in St. Andrew Bay,Florida

Pterospora floridiensis, a new species of acephaline gregarine from the body cavity of the bamboo worm Axiothella mucosa (Polychaeta: Maldanidae), is described. The gamont stage is distinctive and possesses a central cytoplasmic mass and two elongate trunks that bifurcate repeatedly and comprise approximately 60% of the total cell length. The gamont averages 198 (50–545) × 71 (25–180) m (N= 45, 43) from the tip of the trunks to the anterior (the junctional site with the other gamont in syzygy). Gametocysts average 402 (297–545) × 304 (149–495) m (N= 37). The oöcysts measure 22.5 (20.5–23.5) × 8.3 (7.0–10.0) m (N= 30) and possess an internal capsule (average length = 13.9 m, N= 30) containing the sporozoites and aliform wings on the epispore.     

Metal complexes of the schiff bases 2-pyridinylhydrazine and 2-quinolinyl-hydrazine with 2-pyridinecarboxaldehyde N-oxide,including some N-oxide-bridged antiferromagnetic complexes of cobalt(II) and nickel(II)

Metal complexes of 2-pyridine carboxaldehyde 2′-pyridinylhydrazone 1-oxide (poph) and 2-pyridinecarboxaldehyde 2′-quinolinylhydrazone 1-oxide (poqh) are reported with copper(II), nickel(II), cobalt(II), iron(II) and manganese(II). Each ligand appears to function as an ONN donor, via the pyridine N-oxide oxygen, the imine nitrogen, and a pyridine or quinoline nitrogen. The complexes have been characterised by magnetic susceptibility measurements to liquid nitrogen temperature, and also by electronic, infrared, X-ray powder diffraction, and Mössbauer spectra. No magnetic interaction was detected with the copper(II) complexes. All the complexes of metal nitrates appear to be monomers.The complexes of poph with the halides and thiocyanates of nickel(II) and cobalt(II) appear to be six-coordinate and N-oxide-bridged; they exhibit varying degress of antiferromagnetic interaction and the magnetic data for the nickel(II) complexes have been fitted to various models. In contrast, the bulky ligand poqh produces halide-bridged six-coordinate nickel(II) complexes and monomeric five-coordinate cobalt(II) complexes.This behaviour by poqh resembles that of the related NNN ligands paphy and paqhy, which are the Schiff bases of 2-pyridinecarboxaldehyde with 2-pyridinylhydrazine and 2-quinolinylhydrazine, respectively.     

The fine structure of the phoront of Gymnodinioides pacifica, a ciliated protozoan (Ciliophora, Apostomatida) from euphausiids of the Northeastern Pacific

Phoretic stages of the exuviotrophic apostome Gymnodinioides pacifica were examined using transmission and scanning electron microscopy (TEM and SEM). TEM revealed that the mature cyst wall possesses 2 or 3 layers differing by the presence or absence of the third inner layer. This inner layer may represent a different form of the middle wall material. The inner cyst layer is approximately 0.15 microm thick and has striations with a periodicity of approximately 19 nm. The middle cyst layer has a variable thickness and the outer dense layer is approximately 0.1 microm thick. The 3 layered cyst wall had a thickness of 0.3-0.7 microm and averaged 0.5 microm. Advanced phoront stages were enclosed by fully formed cyst walls or by cyst walls thinned to approximately 0.1 microm, as the phoronts prepared to excyst prior to host ecdysis. Additionally, we report the fine structure of the rosette, trichocysts, nuclei, food plaquettes, oral fiber, and other cytoplasmic inclusions. SEM revealed an outer cyst wall layer connected to the secreted peduncle material, which was observed to extend over a wide (15 microm) area on the host setae. Cysts were usually attached at their posterior ends or, less frequently, along their side.     

FANCI is a second monoubiquitinated member of the Fanconi anemia pathway

Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.     

Molecular Cloning and Characterization of a Lobster Gαs Protein Expressed in Neurons of Olfactory Organ and Brain

Abstract: We have isolated from an American lobster ( Homarus americanus ) olfactory organ cDNA library a clone, lobGα_s, with >70% identity to mammalian and arthropod Gα_s sequences. In genomic Southern blots, a fragment of lobGα_s detected only one band, suggesting the lobsters have a single Gα_s gene. In brain and olfactory organ, lobGα_s mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. Gα_s protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobGα_s plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobGα_s mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobGα_s may mediate olfactory transduction. That virtually all ORNs express lobGα_s mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.