Antibodies to HTLV-1 in saliva of seropositive individuals from Japan

Secretory antibodies protect mucosal surfaces against transmission of many viruses. Human T-lymphotropic Virus, Type I (HTLV-I) is transmitted via blood products and via sexual contact across mucosal surfaces. We investigated the presence of HTLV-I-specific antibodies in whole saliva samples from 10 seronegative and 28 seropositive volunteers from a hospital in southern Japan. Antibodies directed to HTLV-I antigens were found in the salivas from 22 of 28 (79%) of the seropositive subjects. None of the seronegative individuals showed evidence of salivary antibodies. Antibodies directed to the envelope antigens of the virus were found in 21 of 22 positive saliva samples. Secretory antibodies may be important in preventing mucosal transmission.     

Influence of adenosine on the stimulatory effect of isoprenaline and insulin on myocardial contractility in vivo.

STUDY OBJECTIVE--In vitro investigations have indicated that adenosine can inhibit beta adrenergic stimulated increases in cardiac contractility. The present study was designed to determine the ability of adenosine to inhibit isoprenaline induced increases in contractility in vivo. Adenosine has been reported to exert its inhibitory effects on contractility by inhibiting adenylate cyclase. Thus, adenosine should have no effect on positive inotropic agents that act independently of adenylate cyclase. We therefore assessed the ability of this nucleoside to inhibit the positive inotropic effect of insulin, a hormone that exerts a positive inotropic effect independently of alterations in cyclic AMP. DESIGN--Saline or adenosine (10 mumol.ml-1) was infused into the circumflex artery at 1 ml.min-1 as a background. Isoprenaline (20 or 200 pmol.min-1) was infused into the artery during saline or adenosine infusion. The response to insulin was determined during hyperinsulinaemic euglycaemic clamp. SUBJECTS--16 adult mongrel dogs were anaesthetised with pentobarbitone. Five dogs were used in isoprenaline studies, and 11 dogs in insulin studies. MEASUREMENTS AND MAIN RESULTS--Dogs were instrumented to obtain measurements of mean arterial blood pressure, heart rate, circumflex artery blood flow (Q), instantaneous left ventricular pressure, and posterior left ventricular wall thickness. We used the slope of the end systolic pressure-dimension relationship (Ees) as an index of myocardial contractility, previously shown to reflect changes in myocardial inotropic state independent of influence from afterload and preload. Left ventricular dP/dtmax was derived from left ventricular pressure with respect to time, and Ees was determined from left ventricular pressure and wall thickness. Neither adenosine, isoprenaline, nor insulin alone caused any significant changes in mean arterial pressure or heart rate. Adenosine caused a significant increase in Q. Both left ventricular dP/dtmax and Ees were significantly increased by either insulin or both doses of isoprenaline. Adenosine inhibited the increases in these indices caused by isoprenaline, but not those caused by insulin. CONCLUSIONS--Adenosine is capable of inhibiting the positive inotropic effect of isoprenaline in vivo. The results suggest that adenosine does not inhibit positive inotropic responses that act independently of the stimulation of adenylate cyclase.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Effect of denervation on the endogenous phosphorylating activity in the cytosol of rat skeletal muscle

An endogenous phosphorylating activity is demonstrated in the cytosol from soleus muscle of the rat which is markedly stimulated after severing the motor nerve fibers to this muscle. The [γ-³²P]ATP phosphotransferase reaction is heat-labile, dependent upon Mg²⁺ but not Ca²⁺ or cyclic GMP, inhibited by a cyclic AMP dependent protein kinase inhibitor, and directly related to the amount of cytosolic protein which provides the endogenous source of both the protein kinase enzyme, ATP, cyclic AMP and phosphorylatable protein substrate. The time-course of the delayed transitory stimulation of the cytosolic phosphorylating activity of the denervated soleus may involve neurotropic factors.