Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes

Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.     

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.     

Should colonoscopy be the first investigation for colonic disease?

Many patients with suspected colonic disease undergo rigid sigmoidoscopy, barium enema examination, and ultimately total colonoscopy, but the need for preliminary radiology has not been formally assessed. A total of 168 patients requiring large bowel investigation were therefore randomised to undergo either rigid sigmoidoscopy plus double contrast barium enema examination or total colonoscopy. Disease was found in 56 patients, including 14 with a carcinoma, 11 with polyps, and 16 with inflammatory bowel disease, the remainder having diverticular disease alone. Of the 89 patients allocated to double contrast barium enema examination, nine required a subsequent colonoscopy for suspected tumour or polyps, three because of incomplete radiological examination, and 12 for rectal bleeding for which no cause was found at the radiological examination. In 16 patients this yielded further information or altered treatment. Of the 79 patients undergoing total colonoscopy, only six required subsequent radiology.As both procedures were well tolerated with no major complications total colonoscopy may be the preferred initial investigation where facilities allow.     

Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.     

Multiple variants in subtelomeric regions of normal karyotypes

We describe a human genomic cosmid clone, 56.1.1, that contains subtelomeric sequences present on multiple human chromosomes. In particular, using fluorescence in situ hybridization, we have identified 16 sites of hybridization on 12 chromosomes. In a sample of 8 unrelated individuals, 10 of these sites showed interindividual variation. Co-hybridization with other polymorphic probes allowed us to demonstrate cytologically heterozygosity at three sites in six individuals. The chromosomal distribution of hybridization sites in a family strongly suggests that these variants are inherited in a Mendelian fashion. These data show that subtelomeric repeats are a rich source of genetic variability. Possible mechanisms of generation of such variants are discussed.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

A review of the USAF/NASA proton bioeffects project: rationale and acute effects.

Ionizing radiation represented one of the important hazards facing the first manned lunar mission. A combined USAF/NASA project was conducted from 1963 through 1969 to estimate the relative biological effectiveness (RBE) of the radiations of space. Approximately 2000 primates (Macaca mulatta) and 5000 mice were irradiated with protons and electromagnetic radiations. The proton energies studied were selected to be representative of the proton spectrum in space. Much of the project was concerned with the use of cyclotrons for proton irradiations and with dosimetry. Biological measurements included clinical findings, physiological changes, hematological changes, histopathology, and mortality. When allowance was made for variation of response as a consequence of depth-dose distribution, the RBE for protons was approximately 1. This was anticipated from earlier theoretical studies and radiation therapy in humans with high-energy charged-particle beams.     

Sporocysts isolated from the southern copperhead (Agkistrodon contortrix contortrix) produce Sarcocystis montanaensis-like sarcocysts in prairie voles (Microtus ochrogaster)

Sporulated oocysts and free sporocysts of a Sarcocystis sp. were isolated from the feces of a southern copperhead (Agkistrodon contortrix contortrix) collected in Arkansas (USA). Twenty sporocysts measured 11.2 by 8.5 microns, lacked a Stieda body, and had four sporozoites and a granular sporocyst residuum. Sarcocysts similar to those of Sarcocystis montanaensis were present in the tongues of prairie voles (Microtus ochrogaster) inoculated orally with 800 sporocysts 128 days previously. Sarcocysts were thin-walled, divided into compartments by septa, and had electron dense projections (0.14 microns) on the primary cyst wall. Infection was not pathogenic for prairie voles under the conditions of this study. No infections were observed in ICR strain laboratory mice (Mus musculus) or white-footed mice (Peromyscus leucopus) following oral inoculation of 800 sporocysts.     

The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth.

Tissue and urokinase-type plasminogen activators are serine proteases with highly restricted specificity, their best characterised role being to release the broad specificity protease plasmin from inactive plasminogen. It has frequently been suggested that these, and similar proteases, are involved in axonal growth and tissue remodelling associated with neural development. To help define what this role might be, we have studied the expression of the plasminogen activators in developing rat nervous tissue. Urokinase-type plasminogen activator mRNA is strongly expressed by many classes of neurons in peripheral and central nervous system. We have analysed its appearance in spinal cord and sensory ganglia, and found the mRNA is detectable by in situ hybridisation very early in neuronal development (by embryonic day 12.5), at a stage compatible with it playing a role in axonal or dendritic growth. Tissue plasminogen activator mRNA, on the other hand, is expressed only by cells of the floor plate in the developing nervous system, from embryonic day 10.5 and thereafter. Immunohistochemical and enzymatic analysis showed that active tissue plasminogen activator is produced by, and retained within, the floor plate. A mechanism is suggested by which high levels of tissue plasminogen activator produced by the stationary cells of the floor plate could influence the direction of growth of commissural axons as they pass through this midline structure.