Absence of “epitheloid cells” in AV-anastomoses (AVA) in rabbit auricular tissue prepared for electron microscopy

Summary AV-anastomoses were studied in rabbit auricles. After perfusion-fixation with glutaraldehyde and post-osmication AV-anastomoses are to be identified only when serial sections are analyzed. Epitheloid cells were not seen in the walls of the anastomosing segments.     

Molecular clock of silent substitution: At least six-fold preponderance of silent changes in mitochondrial genes over those in nuclear genes

Summary For each of eleven different types of nuclear genes, comparisons of the protein coding sequences were made between human, mouse and rat pairwisely, and the evolutionary rate of silent substitution, v _S ^nucl. , was estimated. It is shown that the v _S ^nucl. is not only very high (=5.37×10^–9/site/yr), but also approximately uniform for different genes regardless of the types, which confirms our previous results (Miyata et al. 1980b). This is in sharp contrast to the rate of protein evolution which differes greatly from protein to protein. Furthermore the v _S ^nucl. is shown to be approximately constant with respect to different divergence times, at least within a short time period (75 Myr). Based on these observations, we propose a new molecular clock which has several advantages over a protein clock. Using this clock, we show that the rate of amino acid replacement in the immunoglobulin Ck gene of b4 rabbit is unexpectedly high, almost comparable to the rate of silent changes. This rate may be the highest one for protein evolution that we know so far. We further examine the rate of silent substitutions in mitochondrial genes comparing mouse and rat. Surprisingly the rate is extremely high (35×10^–9/site/yr), at least 6-times as high as the corresponding rate of nuclear genes. Based on the estimate, we discuss a possible origin of the rapid rate found in mitochondrial DNA.     

Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig,rabbit and rat

Summary The number of quinacrine-fluorescent nerve cell bodies and the percentage of the ganglion area occupied by this fluorescence within stretch preparations of the myenteric plexus of the stomach and ileum of the guineapig, rabbit and rat were assessed. The number of quinacrine-positive cell bodies per cm² of plexus varied between 1045 in the rabbit ileum to 2633 in the rat stomach, whilst the percentage of the ganglionic area occupied by fluorescence was approximately 10 %. The distribution of quinacrine-fluorescent nerve fibres and cell bodies in the myenteric plexus was compared to the distribution of nerves revealed by catecholamine fluorescence and by staining for acetylcholinesterase in the stomach and ileum of all three species. Quinacrine fluorescence appears to be selective for non-adrenergic, non-cholinergic nerves; the possibility that it binds to high levels of ATP is discussed.     

Equilibrium centrifugation of measles virus

Measles virus has been centrifuged on different density gradients. It sediments at densities of 1,20 g/cm³ in K-tartrate, of 1,18–1,21 g/cm³ in sucrose, 1,19–1,23 g/cm³ in CsCl and 1,19 g/cm³ in metrizamide gradients. Metrizamide reduced measles virus infectivity. In sucrose gradients sometimes more than one infectious peak was observed. Control Vero cells produced particles of the same densities as measles virus peaks. These peaks did contain actin as the major protein. The relevance of this finding in relation to the presence of actin in measles virus is discussed.     

Antimicrobial defense mechanisms in the horseshoe crab,Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 mm) concentrations of the monovalent cations Na⁺ and K⁺. The protective effect of Na⁺ was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na⁺ (100 mm) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species (Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na⁺ to reflect levels in L. polyphemus blood (400 to 500 mm) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na⁺, obviating the inhibitory effect of Na⁺ on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na⁺ concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na⁺ inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.     

Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern

Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm² and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis. Received: 6 August 1997/Accepted: 8 October 1997     

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands—typically camelid antibodies—that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (K_D ~ 10⁻⁵–10⁻⁶ M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC_10% > 10¹³ vp/mL of resin) and product yields (~50%–80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%–80%), 80- to 400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.     

早期转基因兔胚的培养及外源基因滞留的研究

用显微注射法导入外源Smtpgh基因的早期免胚进行体外培养,并用PCR技术对单个胚中的外源基因进行了检测,以探讨SMTPGH基因在早期免胚中的滞留情况。研究结果表明早期转基因免胚在Tcl99+10％Fcs培养液中有75％发育到囊胚期;其外源基因在8细胞期前没有丢失,随后有逐渐丢失的现象。到胚胎的发育后期,其外源基因的滞留率接近于整合率。     

Localization of GABAA receptors in the rabbit retina

The distribution of gamma-aminobutyric acid_A (GABA_A) receptors in the rabbit retina is investigated and compared with the distribution of GABAergic neurons using immunocytochemical methods. Antibodies against the 1, 2/3, and 2 subunits of the GABA_A receptor label subpopulations of bipolar, amacrine and ganglion cells. Double labeling experiments show that the 2 subunit is colocalized with the 1 and the 2/3 subunits in bipolar, amacrine and ganglion cells. Electron microscopy reveals that in the outer plexiform layer, GABA_A receptor immunoreactivity is present on dendrites of cone bipolar cells adjacent to the cone pedicles. Bipolar cell dendrites are also receptor-positive at synapses from interplexiform cells. Some receptor immunoreactivity is found intracellularly in processes of horizontal cells. In the inner plexiform layer, GABA_A receptor immunoreactivity is present on both rod bipolar and cone bipolar axon terminals at putative GABAergic input sites. Amacrine and ganglion cell processes in sublamina a and b are also labeled.     

Internalized gap junctions in ciliary epithelium of rabbit and rat

Summary Transmission electron microscopy was used to examine internalized gap junctions (IGJ) in rabbit and rat ciliary epithelial cells. A prominent feature of all the specimens studied was the presence of different images of IGJ membrane that entrapped a portion of an adjoining cell. We documented and analyzed more than 500 gap junction (GJ) vacuoles and invaginations, the latter comprising less than 20% of all the structures examined. With ten exceptions found in non-pigmented cells, all the IGJ were unidirectionally internalized within the cytoplasm of pigmented epithelial cells. Morphological signs of autophagic degradation of GJ vacuoles were observed. An essential finding was that once a GJ membrane started to invaginate, a lucidation of a part of the protruding cytoplasm occurred; no planar GJ membranes exhibited such an alteration. The present findings suggest that IGJ derived from the epithelium of ciliary processes arise through an invagination-endocytosis mechanism and are degraded autophagically. This phenomenon may be relevant to aqueous humor production.