Practical points in the care of the patient post-thyroid surgery. It is the purpose of this article to discuss the care of the patient after thyroid surgery. Preoperative preparation will be presented, thyroid anatomy reviewed, and surgical interventions identified. PACU care of the patient identifies specific patient priorities and potential postoperative complications.


Instrument development: writing the items. An important step in instrument development is writing the items that are derived from concept analysis and validation. It is also important to determine if the instrument is to be constructed for criterion-referenced or norm-referenced measurement; examples for both are discussed. Also discussed are methods for developing a blueprint for a psychosocial instrument. Additional methods presented are how to construct the items as simple rather than complex sentences, to keep the sentences short and unambiguous, and to construct the items for an eighth-grade reading level to facilitate patient use.


Managers, monkeys, and mind-set.

Managers, monkeys, and mind-set. One of the most difficult things for a manager to learn is effective time management. The challenge seems to be one of learning what tasks really should be done by the manager and what really can and should be delegated to the staff. This article provides some guidance to managers as they make those decisions.


New opportunities for Americans with disabilities. Historically, individuals with disabilities have been isolated and segregated. Physical and psychological barriers left them significantly disadvantaged, politically powerless, and without legal recourse in matters of discrimination. The Americans With Disabilities Act of 1990 is opening doors and promising new opportunities for individuals with disabilities to contribute and participate fully in our society. The author offers an overview of the purpose and major provisions of the act, which compares in scope to the Civil Rights Act of 1964.


[Use of semi-quantitative approaches in the evaluation and classification of potential teratogenicity of industrial poisons]. To provide a consistent teratogenic risk assessment from animals' data, the system described here suggests an evaluation by stages. Based on the expert system from Mattison, the toxic responses of common species of experimental animals are scored. The scores are then multiplied by the ratio of the lowest adult 'toxic dose A, to the lowest developmental toxic dose D (A/D). The products form 3 categories. Based on the reversibility of the developmental effects, the categories are subdivided into classes A B C D. This system should permit an unbiased risk assessment and a comparison of industrial chemicals' prenatal toxicity.


[Toxicity of chloroform and vitamin A status in the rat]. After a chloroform intraperitoneal injection, lactate dehydrogenase, alanine aminotransferase and particularly aspartate aminotransferase serum activities are much more raised in deficient animals. Liver ornithine decarboxylase (ODC) activity normally decreases in rats between the 4th. and the 7th. month after the weaning. In vitamin A deficient animals, basal values of the enzyme activity are lower and the decrease is deeper. But even at month 7, liver sustains a partial capacity of ODC recovery if retinol is fed during 15 days. Chloroform administration strongly enhances liver ODC activity in normal rats. In the deficiency, stimulation is lower in absolute value but relatively higher if referred to basal level. After retinol refeeding, chloroform stimulates enzyme activity to nearly normal values. Vitamin A deficiency impairs obviously liver ODC activity and its response to chloroform stimulation in rats, but the stroke is at least partially reversible in our conditions. Moreover, deficient animals maintain a non negligible capacity of ODC response under chloroform stimulation.


[Teratogenic action of alpha-asarone in the mouse]. The embryotoxicity and teratogenicity of alpha-asarone were investigated in mice. The drug was dissolved in corn oil, and administered daily, by gavage, on days 6 to 15 of gestation, at 0 (controls), 5, 15, 30 and 60 mg/kg. Fetuses were removed on day 18 by caesarean section and examined using routine teratological methods. A significant maternal toxicity was observed in dams given 60 mg/kg, as indicated by a reduced weight gain. An embryolethality was observed in 15, 30 and 60 mg/kg treated groups. In addition, the highest dose induced fetal malformations, mainly represented by hydrocephaly, extra-ribs, clubfeet and cleft lips.


[Effects of environmental tobacco smoke on prenatal development. (Review of the medical literature)]. For near thirty years, epidemiological studies have coped with the search of possible noxious consequences of an involuntary exposure of pregnant women to environmental tobacco smoke on the gestation and the intrauterine development of embryo and foetus. These studies were mainly retrospectives; a careful study of the methods used (questionnaires, evaluation of exposure, and so on ...) gives evidence that they can rarely avoid serious criticism. As possible effects of intrauterine exposure to environmental tobacco smoke, low birth weight and impairing of other body parameters, perinatal mortality, frequency of abnormalities have been reviewed. As a potential cause, the role of the father tobacco smoking has been especially examined. As a whole, the consequences of a prenatal exposure to environmental tobacco smoke are an extremely controversial subject and no obvious effect has yet been universally recognized. The critical analysis of the studies has shown that, frequently, the epidemiological studies have been interpreted in order to find links between and involuntary exposure to tobacco smoke and some troubles of reproduction, particularly in offspring. As a matter of facts, fundamentally, the noted actually correlations, even if they are statistically significant, are not able to move such links. They are only able to indicate the existence of an association and only, if the eventual role of confounding factors has been properly treated. An interesting case is the potential effects of the father's tobacco smoking. The hypotheses emerging from these examined inquiries remain to be more precisely defined and thoroughly by new studies, preferentially prospective, and, when necessary, completed by animal experiments. It is suggested that a special effort shall be applied to the measurement of the exposure of pregnant women to define toxic compounds originating from environmental tobacco smoke. Presently, it is not possible to draw a conclusion on the noxious or innoxious influence of the involuntary exposure of pregnant women to environmental tobacco smoke, particularly as far as potential risks for foetus are concerned.


An assessment of proteolytic enzymes in Tetrahymena thermophila. Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.


Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences. A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.


Advanced Trauma Life Support aboard RFA Argus. The Advanced Trauma Life Support (ATLS) system was adopted for casualty reception and resuscitation. ATLS permitted well-informed triage decisions to be made, coupled with appropriate initial, possibly life-saving, treatment. The training given on board has continued to benefit patients treated by ex-Argus staff in their peacetime roles.


Electron microscopic analysis of circumsporozoite protein trail formation by gliding malaria sporozoites. Immunoelectron microscopic techniques were utilized to characterize the morphology of circumsporozoite protein-containing trails deposited on various substrates by gliding Plasmodium berghei and Plasmodium falciparum sporozoites. The basic components of the trails are beadlike particles, 25 to 90 nm in diameter, which are devoid of unit membrane and have an electron-lucent center. Trails were captured on formvar-covered grids coated with anticircumsporozoite protein monoclonal antibodies and compared with trails produced on uncoated formvar; the results suggest that material containing circumsporozoite protein forms the matrix within which the particles are embedded. The trails exhibit morphological features similar to those displayed by circumsporozoite precipitation reactions; of note is the demonstration of sheaths of circumsporozoite protein-containing material that emanate from sporozoites prior to their gliding. The sheaths narrow into accumulations of electron-dense material, which eventually taper to form typical trails. The structural manifestation of sheaths and other morphological details of the formed trails enables us to correlate sporozoite behavior during trail formation with the motile actions of gliding sporozoites observed by light microscopy.


Sporogonic development of Leucocytozoon smithi. The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.


Meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor. Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.


Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies. Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.


Discontinuous genetic variation among mesophilic Naegleria isolates: further evidence that N. gruberi is not a single species. Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species--that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica. Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristics temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.


Experimental horizontal transmission of Enterocytozoon salmonis to chinook salmon, Oncorhynchus tshawytscha. Enterocytozoon salmonis was transmitted to chinook salmon Oncorhynchus tshawytscha by feeding tissues infected with the parasite and by cohabitation of noninfected fish with experimentally infected fish. Affected fish (dead and survivors) in both transmission trials had gross and microscopic signs of the disease and merogonic and sporogonic stages of the parasite. There were no morbidities or mortalities, or evidence of the parasite among control fish in either study. Results suggest that the parasite may be contracted by indirect contact among healthy and infected fish held in crowded ponds or net pens or by direct ingestion of spores found in the water.


Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins. We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors.


Dura and cranial base reconstruction by external oblique fascia and rectus abdominis muscle flap. The rectus abdominis muscle flap, combined with the external oblique fascia, were utilized in cranial-base reconstruction. In a wide defect caused by resection of a giant meningioma, the dura was reconstructed with the external oblique fascia and anterior sheath, and the cranial-base defect was reconstructed with the rectus abdominis muscle flap.


Reconstruction of recurrent pressure sores using free flaps. The authors describe two successful reconstructions of recurrent pressure sores with free fasciocutaneous flaps. In Case 1, a free lateral thigh flap pedicled on the first and third direct cutaneous branches of the deep femoral vessels was used to cover a large recurrent sacral pressure sore. The vascular pedicle was dissected to the deep femoral trunk proximally and anastomosed to the inferior gluteal vessels. In Case 2, a free medial plantar flap was transferred to a recurrent ischial pressure sore. The vascular pedicle was dissected to the posterior tibial vessels proximally. The long vascular pedicle of the flap was passed through the femoral subcutaneous tunnel, and end-to-side microvascular anastomoses were performed to the superficial femoral trunk without any vein grafts. The authors advocate the use of free tissue transfer for recurrent pressure sore reconstruction.


Cryopreserved allogeneic vessel and nerve grafts: hind-limb replantation model in the rat. The authors have previously reported the permanent storage of skin and cutaneous flaps using cryopreservation. In this study, replantation of the hind limb was performed in rats, using an allogeneic femoral artery, vein, and sciatic nerve which had been cryopreserved for more than 3 weeks. Femoral arteries, veins of 2 cm in length, and sciatic nerves of 1.5 cm in length from Lewis rats were cryopreserved for more than 3 weeks to decrease antigenicity. The hind limb of a Brown Norway rat was completely amputated at its mid-length and the cryopreserved artery, vein, and nerve were interposed in the anastomosis of the femoral artery, vein, and sciatic nerve. Immunosuppressants were not used. Hind limbs were replanted in six rats, and all cases survived. Toe spread and gait-foot analysis revealed good results at 3 months postoperatively. Excellent axonal regeneration was observed in the pathologic examination of the sciatic nerve 1 cm distal to the transfer. This report introduces a long-term storage method for vessels and nerves and extends the possibilities for clinical use of cryopreserved vessels and nerves, using replantation in a rat model.


Successful replantation of the lower leg after 42-hour ischemia: case report. A case of right lower-leg replantation after 42-hr ischemia is presented. Revascularization of the other foot with circulatory decompensation after 36-hr ischemia was carried out simultaneously. The replanted lower leg survived. Following its shortening in replantation by 12 cm, right lower-leg lengthening by 8 cm was carried out 1.5 years after replantation with the aid of a distraction apparatus. The locomotor function in both lower extremities recovered.


Successful digital replantation after 42 hours of warm ischemia. Previously, 6 to 10 hr were believed to be an acceptable limit of warm ischemia for successful digital replantation. The longest warm ischemia time ever reported was 33 hr. This report presents successful replantations of two fingers after 42 hr of warm ischemia.


Secondary coverage of the hand using a dorsalis pedis plus first web space free flap. Traditionally, severe degloving injuries of the hand have been treated with random abdominal or pedicled groin flaps, which offer good cutaneous coverage but do not provide sensibility. The authors present the results of the application of an extended dorsalis pedis plus first web space of the foot flap to resurface the hands of five male patients who had been treated originally with random abdominal or pedicled groin flaps. The reported flap has the advantage of providing the patient with up to three different nerve territories, aiding in a better functional use of the hand.


Reinnervated radial forearm free flaps in head and neck reconstruction. The radial forearm flap has proved to be a reliable free flap for intraoral reconstruction after major head and neck ablative surgery for cancer. In contrast to the myocutaneous flap, it is thin and flexible, and as a result, it is better suited to conforming to the irregular surface which remains over an intact or restored mandible. A criticism of both techniques however, is that while the flap effectively fills the defect, it serves as an insensate reservoir in which food and saliva can collect. A modification of the reinnervated radial forearm free flap is presented, with discussion of its use in three patients, following extensive resection of the floor of the mouth and tongue.


Morphologic characteristics of muscles grafted in rabbits with neurovascular repair. In 34 female white rabbits, rectus femoris (RFM) muscles were grafted with immediate anastomoses of the vasculature (VA) and with nerves either left intact (NI-VA) or with nerves repaired (NR-VA). The purpose of the study was to compare the morphologic changes that occur in NI-VA grafts and NR-VA grafts from 8 to 120 days after grafting. After 8 days, nearly complete survival of all muscle fibers was found. The muscle mass and single fiber cross-sectional area (CSA) of the NI-VA group remained near control values for the first 30 days and then declined to 82 percent and 62 percent of the control values, respectively. Little evidence of morphologic disruption was observed. The NR-VA grafts displayed a significant denervation atrophy within the first 15 days, with relative values 67 percent of the control value for mean mass and 53 percent for single fiber CSA. By 120 days, mass and CSA recovered to 80 percent and 62 percent of control values. Although the similarity of the deficits in the nerve-intact and nerve-repaired grafts suggest that tenotomy and repair, rather than innervation, were the major limitations, the mechanism was not resolved.


A simple cuff-suture technique for microvascular anastomosis. The use of synthetic cuffs to simplify and hasten microvascular anastomoses is offered as an alternative to conventional methods. In this study, a soft, non-irritating, silicone rubber cuff was used to construct end-to-end and end-to-side cuff-sutured anastomoses in rats. This technique was as rapid as other non-sutured anastomoses, required no assistant, and provoked no foreign-body reactions.


Leukocyte adhesion to vascular endothelium. Leukocyte adhesion and emigration are controlled by soluble mediators and effected by various adhesion molecules. Currently, three major families of adhesion receptors are known to contribute to this process: integrins, vascular selectins, and immunoglobulin-like receptors. These adhesion systems are not additive and mutually replaceable, but appear to constitute a cascade of events. Leukocyte margination is followed by rolling, firm adhesion, emigration, and migration in the interstitial space. In addition, biomechanical parameters like leukocyte deformability and shear stress exerted by the flowing blood modulate the efficacy of adhesive interaction. This article briefly reviews the molecular nature, biologic regulation, and physiologic function of pertinent adhesion receptors.


Smoking impairs the response to a physical training regime: a study of officer cadets. One hundred and sixty five officer cadets completed a 6 month physical training programme; 47 (28%) were smokers and 118 (72%) were not. Improvements in fitness were measured by the Army Personal Fitness Assessment (APFA) which scores for both strength and endurance. Initially both groups had similar APFA scores [mean (SEM)]: smokers 114 (3.1), non smokers 120 (2.3), difference not significant. Six months later both groups had improved their scores: smokers 131 (2.7), non smokers 143 (2.3), but the smokers were now significantly less fit (p < 0.01). The British Army needs a more effective anti smoking policy as 50% of young soldiers continue to smoke, and have an increased risk of premature ischaemic heart disease when compared with their civilian counterparts. Physical fitness remains important in the Army and evidence such as this may help persuade soldiers to give up smoking before standard tests of cardiovascular or lung function show any abnormalities, with benefits for the future health of the Army.


A new chart to assist with advanced trauma life support. Many studies have drawn attention to deficiencies in the management of major trauma, both in the UK and elsewhere. One area that has received little attention is the documentation of such cases in the Emergency Room. When outcome may be sub-optimal, documentation assumes greater importance if advances are to be made in the organisation of trauma care. Based upon the American College of Surgeons Advanced Trauma Life Support (ATLS) protocols, the authors have designed a document that records dynamically what happens to the multiply injured victim on arrival in the Emergency Room. It unifies the recording of vital signs, whilst acting as an assessment and resuscitation template. By ensuring no life-threatening illness is missed it is likely to improve patient survival. The document can act as a basis for teaching and a medico-legal record, whilst providing the necessary data for quality assurance and outcome audit.


Chronic pancreatitis in the British Army 1978-1989. A retrospective study was made of all 28 soldiers with chronic pancreatitis first diagnosed between 1978 and 1989. All patients were male, alcohol was the aetiological factor in 90%, the mean age at diagnosis was 30 and the commonest mode of presentation was with recurrent painful episodes of pancreatitis. Endocrine and exocrine pancreatic insufficiency occurred in a quarter and a third of patients respectively and one third required surgical intervention.


A case of factitious fever and 'epilepsy'. We describe the case of a 23 year old male who presented with a history of intermittent pyrexia associated with apparent episodic loss of consciousness. During these events, thermometers placed in the rectum and axillae supported the elevated oral temperature readings. After numerous investigations including electroencephalography had excluded organic disease, the patient was observed applying a hot douche to his rectum prior to his temperature being recorded.


Complications of prophylactic intercostal tube drainage--including tension pneumothorax. A case is presented of tension pneumothorax associated with intercostal tube drainage. Complications of intercostal tube drainage are reviewed.


The corps disease: brucellosis and its historical association with the Royal Army Medical Corps. Brucellosis (also known as Malta, Mediterranean or Undulant Fever) has aptly been nicknamed the Corps Disease because of the major role played by the Royal Army Medical Corps in elucidating its nature and discovering its mode of spread, thus leading to its prevention and eradication. This history of brucellosis, incorporating a complete bibliography of all references to the disease in the Journal of the Royal Army Medical Corps from 1903 to 1992, documents the fascinating story of this association.


The number of cells used for immunotherapy of repeated spontaneous abortion influences pregnancy outcome. One hundred and sixty-eight women were immunized on a single occasion with paternal mononuclear cells (MNC) for recurrent spontaneous abortion (RSA) and pregnancy outcomes were analysed with respect to the number of MNC given. The study was done in a prospective sequential fashion using all MNC recoverable from a unit of the spouse's blood and both patients and investigators were blinded as to the number of cells injected. Women receiving low and mid-range doses of MNC (58-305 x 10(6) and 308-567 x 10(6), respectively) had a significantly higher pregnancy success rate (57%) than those receiving the high (568-2677 x 10(6)) dose of MNC (41%). In 77 consecutive patients the diameter of the largest immediate skin flare reaction at the site of subcutaneous injection was recorded. No correlation was found between the skin flare response and the number of MNC injected. Our data suggest that a blinded trial of paternal MNC immunization comparing what appears to be optimum numbers of cells (100-550 million) to a low dose inoculum (e.g., 10 million), again noting the sizes of the skin flare reactions, might answer questions about efficacy and placebo effects of immunotherapy for RSA.


Differential distribution of interleukin-1 alpha and interleukin-1 beta proteins in human placentas. Interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were identified in samples of first trimester and term placentas by immunocytochemistry using two sets of polyclonal antibodies to recombinant IL-1. All samples contained the two species of IL-1, which predominated in different types of cells: IL-1 alpha was contained primarily in placental villous trophoblastic and stromal cells whereas IL-1 beta was localized to cells in fetal and maternal blood. With respect to the cellular localization of the two proteins, early and late gestation tissues were similar. Although these results were generally consistent for the two sets of antibodies, differences were noted in the binding patterns of the individual reagents. The findings in this study provide strong support for the postulate that IL-1 modulates placental development and suggest that IL-1 alpha may be the preferred species in extraembryonic tissues.


Distribution of lymphocyte subsets in rat milk from normal and Trichinella spiralis-infected rats. We have shown that T. spiralis-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. This study addresses the change of lymphocyte populations in rat milk during normal and disease conditions. Two color flow cytometric analysis was performed for milk lymphocytes. T cells (OX19+) made up 45% of rat milk lymphocyte population. T helper cells (Th) composed 35% of total T cells while T cytotoxic/suppressor (Tcs) cells constituted 34%, giving a Th/Tcs ratio of 1.03. The corresponding ratio Th/Tcs in peripheral blood was 2.8. Approximately 21% of OX8+ cells in rat milk were OX19- natural killer (NK) cells. When using the monoclonal antibody 3.2.3 (NKR-P1), 43% of lymphocytes in control rat milk and 14% of blood lymphocytes were NK cells. This indicates a selective passage of these cells into milk. In T. spiralis-immunized rats, the percentage of total T cells was slightly decreased; however, Th and Tcs cells were consistent as compared to control milk. The percentage of NK cells (OX8+OX19-) in milk from T. spiralis-immunized rats was significantly higher than that from control milk (65% vs. 21%, respectively, P less than 0.01). This result was confirmed using the monoclonal antibody 3.2.3 which showed that milk from immunized rats contained 63% NK cells compared to 43% in normal milk (P less than 0.01). This study suggests that NK cells are selectively passaged into rat milk and T. spiralis infection induces an increase of NK cells in milk.


Antifertility effects of antisperm cell-mediated immunity in mice. C57BL/6 female mice were immunized with allogeneic (DBA/2) sperm in Freund's adjuvant either subcutaneously (s.c.), transcervically into the uterine lumen (i.u.), or with a combination of s.c. and i.u. immunization approaches. Control mice received DBA/2 lymphocytes, human erythrocytes or saline in adjuvant using the same immunization protocols. Immunization with sperm or control cells in adjuvant exclusively by s.c. or i.u. approaches did not affect subsequent fertility, although sperm-injected mice from both protocols had high titers of circulating antisperm antibodies. In contrast, mice that were immunized with sperm in adjuvant by a combination of s.c. and i.u. injections demonstrated significant reductions in fertilization rate and number of viable fetuses and an increased rate of fetal resorption when compared with non-immunized and control-immunized mice. Mice receiving sperm by the s.c./i.u. protocol had high titers of antisperm antibodies and a marked infiltration of T lymphocytes and macrophages into the uterine endometrium. To determine whether cellular immune mechanisms contributed to the infertility effect, T lymphocytes from spleens and pelvic lymph nodes of s.c./i.u. sperm-immunized mice and non-immunized mice were passively transferred to naive syngeneic female recipients which were subsequently mated. The total number of fetuses on day 15 of pregnancy was significantly reduced in mice receiving T-lymphocytes from sperm-immunized mice and a significant increase in fetal resorption sites was also observed. These mice did not have detectable titers of circulating antisperm antibodies, but had a significant infiltration of CD4+ T lymphocytes and macrophages in the uterine epithelium and endometrium. These data indicate that intrauterine antisperm cell-mediated immunity can be induced in mice by a combination of systemic and intrauterine immunizations and provide evidence for the existence of reproductive tract mucosal antisperm cellular immune responses that adversely affect fertility and pregnancy.


Alloimmune neonatal neutropenia is a potential side effect of immunization with leukocytes in women with recurrent spontaneous abortions. Alloimmunization of a mother against granulocytes causing alloimmune neonatal neutropenia (ANN) in her newborn was found likely to be attributed to previous intradermal injections of paternal lymphocytes. Immunotherapy with leukocytes which was performed for recurrent spontaneous abortions hence provides the possibility of granulocyte alloimmunization and increases the risk of the occurrence of ANN.


Laparoscopic uterine suspension as an adjunctive procedure at the time of laser laparoscopy for the treatment of endometriosis. Uterine suspension has been advocated as an adjunctive procedure at the time of conservative surgery for endometriosis but has seldom been used at the time of CO2 laser laparoscopic treatment of endometriosis. In this study of 225 patients treated for cul-de-sac endometriosis by CO2 laser laparoscopy between 1984 and 1989 uterine suspension was performed as an adjunctive procedure at the time of laparoscopy. The result was a cumulative pregnancy rate of 80.0%. Life-table analysis was performed, and monthly fecundity rates were calculated as 15.58%, 6.29%, 17.86% and 7.89% for Revised American Fertility Society (RAFS) endometriosis stages I to IV respectively. CO2 laser laparoscopy and laparoscopic uterine suspension alleviated preoperative pelvic pain complaints in 94% of the patients. Monthly fecundity rates for RAFS stage I endometriosis, which exceeded previously reported rates following expectant management, medical management and conservative surgery, were attributed to laparoscopic uterine suspension, which had not been previously reported as an adjunct to CO2 laser laparoscopy.


Effect of thrombin-induced hemostasis on the efficacy of an absorbable adhesion barrier. Serosal injury, bleeding and fibrin deposition are major factors in the development of surgical adhesions; meticulous hemostasis is desirable but not always achievable. The effects of thrombin on adhesion formation and the performance of Interceed Barrier were tested in separate series using a standard model and two levels of bleeding: an "oozing" model in which rabbit uterine horns were scraped to produce uncontrolled punctate bleeding and a "bleeding" model, in which four small blood vessels nicked on the ligament to each horn produced heavier bleeding. Substantial clots in the bleeding model were not removed. Adhesions, assessed after two weeks, were not worsened by the use of thrombin to control bleeding. While Interceed Barrier alone did not reduce adhesions at sites of bleeding, achieving hemostasis with thrombin and then applying Interceed Barrier significantly reduced adhesions. The effect was not achieved by applying thrombin to previously blood-soaked Interceed Barrier. The efficacy of Interceed Barrier applied after achieving hemostasis was further improved by moistening it with heparin. Achieving hemostasis at a bleeding site with thrombin facilitates the efficacy of Interceed Barrier.


Clinical and histologic classification of endometriomas. Implications for a mechanism of pathogenesis. One hundred eighty-seven consecutive patients with persistent ovarian cysts and endometriosis underwent laparoscopic evaluation and ovarian cystectomy. All patients had been followed for a minimum of 6 weeks prior to surgery. The cysts were identified initially to be endometriomas based on their gross appearance and the presence of endometriosis at other pelvic sites. Presumed endometriomas were classified into three types based on size, cyst contents, ease of removal of the capsule, adhesions of the cyst to other structures and location of superficial endometrial implants relative to the cyst wall. After clinical laparoscopic classification, the cysts were evaluated histologically without knowledge of the clinical assessment. Histologically small (< 2 cm), superficial ovarian cysts were always endometriomas, and the cyst wall was very difficult to remove (type I). Large cysts with easily removed walls were usually luteal cysts (type II). Large cysts with walls adherent in multiple areas adjacent to superficial endometriosis were generally endometriomas but some also had histologic characteristics of functional (luteal or follicular) cysts (types IIIa and IIIb). These findings led to the conclusion that superficial ovarian endometriosis is similar to endometriosis in extra-ovarian sites in that the formation of superficial cysts is limited in size by fibrosis and scarring. In contrast, large endometriomas may develop as a result of secondary involvement of functional ovarian cysts by the endometriotic process.


Laparoscopic supracervical hysterectomy using a single-umbilical puncture (mini-laparoscopy). Laparoscopic supracervical hysterectomy with bilateral salpingo-oophorectomy utilizing the single-puncture technique (mini-laparoscopy) is presented here for the first time. The surgical outcome of four patients in whom this minimally invasive approach was employed demonstrates the effectiveness of the single-puncture technique as an alternative to the currently used multiple-puncture method for hysterectomy. Laparoscopic supracervical hysterectomy with removal of the uterus and adnexa through the umbilical puncture site is an effective alternative for patients with a healthy cervix, no associated genital prolapse and moderate uterine enlargement. Advantages of this new modality include reduced blood loss and operating time, and the decreased possibility of surgical injury to the ureters, bladder and bowels. The risk of development of malignancy in the retained stump is almost eliminated by the laparoscopic removal of the upper endocervical canal and cauterization of the lower endocervical canal and exocervix.


Determining the time of the urinary luteinizing hormone surge. Does it facilitate the interpretation of endometrial biopsy results? Recent data suggest that endometrial maturation correlates highly with the postovulatory day as determined by ultrasound or identification of the urinary luteinizing hormone (LH) surge. A study was performed to determine the level of correlation between two methods of endometrial biopsy (EB) interpretation. Method 1 correlated histology with the onset of the next menstrual period; method 2 correlated it with the postovulatory day relative to the LH surge. Forty EBs were analyzed. Both methods agreed that 20/40 EBs were in phase and 6/40 EBs were out of phase (greater than a two-day lag between histologic endometrial maturation and chronologic dating). Of the remaining 14 EBs, 8 were in phase by method 1 and out of phase by method 2, and 6 were out of phase by method 1 and in phase by method 2. Thus, 35% of EBs were considered in phase by one method and out of phase by the alternative method. This implies that 35% of management decisions regarding whether to repeat an EB or treat an out-of-phase EB may be influenced by the method of interpreting EBs.


Decreased amniotic fluid index in term pregnancy. Clinical significance. In a study of 331 term pregnancies a four-quadrant technique was used to obtain amniotic fluid index measurements, and the results were compared with the current widely used single-pocket measurement. In contrast to the "2-cm rule," the amniotic fluid index measurements consistently demonstrated higher sensitivity in predicting poor fetal outcome with no decrease in specificity. It was noted that pregnancies with an index of < or = 8 cm showed higher incidences of meconium staining, cesarean delivery for fetal distress, abnormal fetal heart rate monitoring and Apgar scores of < or = 7 or less at one minute.


Fetal weight-cerebellar diameter discordance as an indicator of asymmetrical fetal growth impairment. In this study on the effect of fetal growth impairment < or = 25th percentile on the transverse cerebellar diameter and its relationship with other fetal biometric parameters, the sample comprised 50 women with singleton pregnancies referred for ultrasound evaluation because of clinically suspected intrauterine growth retardation. The cerebellar diameter of asymmetrically growth-impaired fetuses remained within the normal range although it was found to be reduced when compared with that of normal fetuses (4.4 +/- 0.9 versus 4.8 +/- 0.7, P < .05). Although other biometric parameters (biparietal diameter, femur length and abdominal and head circumferences) were also reduced in growth-impaired fetuses, the ratios of these biometric parameters to cerebellar diameter were similar to those of unaffected fetuses. Fetal weight was affected to a greater extent than the cerebellar diameter, leading to discordance between the two parameters. This discordance identified almost all asymmetrically growth-impaired fetuses with a sensitivity of 95.6% and specificity of 96.3%. In contrast, the ratio of head circumference to abdominal circumference remained normal in more than half of the fetuses. Fetal weight-to-cerebellar diameter discordance is a very sensitive and specific indicator of asymmetrical fetal growth impairment.


Inflammatory cell infiltrate in the cervix as a predictor of residual cervical intraepithelial neoplasia after conization. From January 1985 to January 1991, 112 patients underwent conization of the cervix for dysplasia. Twenty-one had incomplete resection, with cervical intraepithelial neoplasia (CIN) extending to the endocervical margins of resection. Only 10 had persistent CIN in follow-up. A decrease in the inflammatory cell infiltrate in the cervical stroma of the conization specimen was associated with persistent disease. A simple technique for quantitating this inflammatory cell infiltrate was used. Smoking was associated with a suppression of this local immune response in the cervix.


Nonstress test assessment of twins.

Nonstress test assessment of twins. Twin pregnancies have higher perinatal morbidity and mortality rates than singleton pregnancies. Researchers have demonstrated that one major benefit of prenatal care in the twin gestation is reduced fetal death rate. This study to determine the relationship of nonstress tests (NSTs) to pregnancy outcome in twin gestations comprised 665 women who delivered at Los Angeles County-University of Southern California Women's Hospital from January 1985 to January 1989. These patients, all of whom had prenatal care (PNC), were subdivided into two groups: (1) PNC and NSTs and (2) PNC and no NSTs. The groups did not differ statistically with regard to gravidity, parity and abortions. NSTs were selectively done on twin gestations complicated by discordancy or other fetal/maternal complications. Ten pregnancies were complicated by fetal demise of one or both twins in patients who received prenatal care without NSTs. Among the NST group there was one fetal demise. Although the NST group had fewer fetal deaths, the reduction was not statistically significant (P = .062). Infant birth weight was identified as a confounder because the NST group had a statistically higher mean birth weight. Definitive proof of the ability of NSTs to reduce the fetal death rate in twin gestations complicated by discordancy or other pregnancy complications awaits a large, prospective, randomized trial.


Epidermal thickness measurements in vaginal intraepithelial neoplasia. A basis for optimal CO2 laser vaporization. The current management of vaginal intraepithelial neoplasia (VAIN) often involves the use of laser vaporization. A study was performed to measure the epithelial thickness in both premenopausal and postmenopausal women with various grades of VAIN to determine the optimum depth of tissue destruction if laser vaporization is used for therapy. Hematoxylin and eosin-stained tissue sections were examined with light microscopy and measurements made with a calibrated micrometer. Sixty-three biopsies from 56 patients were studied. Patients' ages ranged from 22 to 84 years, with a mean of 56. Thirty-six had a prior history of cervical neoplasia. Thirty-nine patients (70%) had VAIN III, 10 had VAIN II, and the remaining 7 patients had VAIN I lesions. The involved epithelium varied from 0.10 to 1.4 mm in thickness, with a mean of 0.46. Noninvolved vaginal epithelium varied in thickness from 0.10 to 0.70 mm, with a mean of 0.28. Koilocytosis was noted in only 9 of the 63 biopsy specimens. In comparing the thickness of involved epithelium in a given patient to that of an adjacent area of normal-appearing epithelium, the epithelium containing VAIN tended to be thicker. The recommended depth of epithelial destruction with laser vaporization in the literature varies widely and appears to have largely an empiric basis. Our study attempted to provide a scientific basis for laser destruction of these lesions. The results obtained indicate that epithelial destruction to a depth of 1.5 mm, including the zone of thermal necrosis, should be sufficient to destroy epithelium containing VAIN without damage to surrounding structures.


Extraovular and intraovular uterine contraction monitoring. A comparison. We tested a new method of monitoring intrauterine contraction pressure. The pressure transducer is simply inserted between the fetal membranes and uterus after checking placental placement with ultrasonography. To evaluate this method, a prospective, randomized study was done to compare intraovular versus extraovular intrauterine contraction monitoring in patients undergoing serial labor induction with oxytocin. Study parameters were length of labor, cesarean section rate, Apgar scores and febrile morbidity rate. Two groups of 32 patients each underwent oxytocin induction for postdatism, diabetes or hypertensive disorders of pregnancy. Our results show that extraovular intrauterine contraction monitoring resulted in a better outcome with respect to all the parameters evaluated. Specifically, it had a shorter induction interval, lower cesarean section rate, lower rate of maternal febrile morbidity and comparable neonatal complications. The technique was safe and easy to learn and apply.


Prophylactic intrapartum amnioinfusion for patients with oligohydramnios. A prospective randomized study. This prospective study evaluated whether prophylactic saline amnioinfusion among patients with amniotic fluid index (AFI) < or = 5.0 cm decreases the incidence of adverse fetal outcomes. Randomization of 53 patients with decreased AFI at term, resulted in 21 patients' receiving prophylactic saline amnioinfusion early in labor, prior to development of an abnormal fetal heart rate tracing. For the treatment group the mean AFI on admission was 3.0 cm, and the postamnioinfusion AFI was 8.9 cm. For 32 comparison (noninfusion) patients, the mean AFI was 2.9 cm; the group consisted of 17 patients randomized to receive no amnioinfusion (control group) and 15 patients who refused to participate in the study. There was no statistically significant difference between the amnioinfused and nonamnioinfused patients with regard to age, parity, gestational age, AFI at admission or duration of first or second stage of labor. Amnioinfusion resulted in no statistically significant reduction in the incidence of recurrent variable decelerations/bradycardia (26.3% vs. 46.6%), intrapartum resuscitation with terbutaline (5.2% vs. 10.0%), cesarean section for fetal distress (9.5% vs. 9.3%), fetal-acidosis (10.5% vs. 12.0%) or Apgar scores < 7 at five minutes (5.2% vs. 0%) in patients with oligohydramnios.


AIDS (HIV) risk assessment in an inner-city women's clinic. Risk assessment and antibody testing are potential modalities through which interventions to reduce heterosexual and perinatal transmission of human immunodeficiency virus (HIV) can occur. More than 98% of women attending inner-city adult gynecology, adult contraception and teen family planning clinics successfully completed self-administered HIV (AIDS) risk assessment questionnaires and received individual counseling, risk-reduction education and referral for antibody testing. Fourteen percent of the women (671/4,802) reported at least one HIV risk factor. Multiple sexual partners and intravenous drug use were the most commonly reported HIV risk factors in this population.


Incidence of salmonellosis in local hens' eggs. Evidence indicates that outbreaks of salmonellosis may be due to infected eggs. This prompted a study on the incidence of salmonella in eggs. This study was done during summer 1991 and 900 eggs were examined in all. None of the eggs examined were found to be infected with salmonella. However, the result obtained should be treated with care and analysed with a critical eye. The result obtained does not imply that local flocks are not infected by salmonella.


Mefenamic acid in prevention of premature labour. Women at risk of premature delivery were divided randomly into 2 groups of 80 each. Mefenamic acid 500mg 3 times a day or a placebo was given in a double blind fashion. Preterm delivery occurred in 15% of the treated group and 40% of the control group (p < 0.005). The mean birth weight in the test group was higher as compared to the controls. There were no cases of foetal malformations in either of the groups. The results support the use of Mefenamic acid in preventing premature labour.


Dietary habits of technical and vocational students in Riyadh, Saudi Arabia--I. Meal skipping. 452 male technical and vocational students aged 16-25 years old were surveyed for their dietary habits at Riyadh, Saudi Arabia. The data presented here cover the habit of meal skipping. Results showed that dinner was skipped by 28%, breakfast by 20% and lunch was skipped by 15%. Differences are discussed according to age cohorts and accommodation status subgroups.


Environmental health and the South African community. The success of an environmental impact assessment programme will depend on the perceptions of a community. There are differences in the opinion of the population groups of South Africa concerning the perceived environmental threats to health.


Lying in children.

Lying in children. Lying is a common problem in children. The causes of lying are numerous. With proper management including good parental role models, minimising or preventing contributing factors, and appropriate discipline, most children outgrow the tendency to lie.


Comparison of the myotonic dystrophy associated CTG repeat in European and Japanese populations. Gene amplification using polymerase chain reaction (PCR) was carried out on DNA samples from a total of 92 normal subjects and 52 subjects with myotonic dystrophy (DM) from European and Japanese populations, to determine the copy number of the CTG repeat associated with DM for each group. In the two populations, the number of repeats on normal chromosomes only were compared, as CTG copy number on DM chromosomes was difficult to determine by PCR alone. In this study, normal chromosomes were found which had as many as 35 copies of the repeat, which is larger than the normal range reported previously but still does not overlap with the repeat number associated with DM pathology, which is at least 50 copies. Using data from normal chromosomes from unrelated subjects, the frequencies of five, 11, and 13 copies of the CTG repeat were found to be significantly different between the two populations, with five and 11 copies more commonly seen in the European population and 13 copies in the Japanese population. This difference may be the result of natural divergence of the normal chromosomes between the population groups.


Minimal expression of myotonic dystrophy: a clinical and molecular analysis. A clinical and molecular study is reported of 83 patients considered to be minimally affected with myotonic dystrophy (DM). These had been identified in three ways: 60 subjects were identified on clinical grounds and were divided into those with and those without neuromuscular involvement (groups I and II); nine subjects were at high risk of carrying the DM gene but had a normal phenotype (group III); and 14 were parents of definitely affected patients where neither parent showed clinical abnormalities (group IV). PCR analysis of the CTG repeat in the DM gene showed a range of 70 to 230 repeats for the younger at risk patients in group III, while the asymptomatic gene carriers in group IV had 53 to 60 repeats. The sensitivity of diagnosis by EMG was found to be 39%. For ophthalmic signs this was 97.5%. This suggests that assignment on the basis of minimal clinical features carries a significant error. Molecular analysis, in conjunction with established clinical investigations, should prove valuable in the identification and exclusion of minimal myotonic dystrophy.


The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy. The gene for myotonic dystrophy (DM) has recently been isolated and amplification of an unstable CTG trinucleotide repeat, located within the DM gene, has been identified in virtually all patients studied to date. A high proportion of DM families who are studied show a progressively earlier age of onset with succeeding generations and, in the few pedigrees reported so far, an increasing degree of amplification of the CTG repeat has been noted to parallel this trend. It has been implicit in several of the original reports on the nature of the changes in the DM gene that knowledge of CTG amplification status at the DM locus of a person will provide useful information concerning prognosis. However, no studies of genotype-phenotype correlation have been reported and there are no specific data on which to base such counsel. In this paper we report the correlation between the degree of CTG amplification and age of onset in 109 DM gene carriers from 17 families. Included are parent-child and sib-sib comparisons which provide a framework in which to incorporate DNA diagnostic studies when counselling subjects and families at risk for DM.


Presymptomatic diagnosis of myotonic dystrophy. The discovery of an expanded (CTG)n repeat sequence in myotonic dystrophy (DM) has greatly improved our ability to detect DM gene carriers who have few or none of the classical signs of this disorder. We report here our experience with two such groups of gene carriers. We used a PCR based protocol that should be especially sensitive to small increases in CTG triplet number which might escape detection by conventional Southern blot analysis. Our analyses show that on 100 non-DM chromosomes the number of CTG triplets ranged from five to 37. We then studied 17 obligate gene carriers aged 55 years and over who showed no muscle weakness. All of the gene carriers in this group showed a relatively small increase in the number of CTG triplets (52 to 90 CTG triplets) with limited somatic mosaicism. We subsequently studied 11 subjects (aged 19 to 36 years) who had previously been identified as gene carriers by genetic linkage studies, but who lacked diagnostic signs. In this prospectively studied group, nine subjects showed an expanded allele, confirming the earlier prediction from linked genetic markers. The other two subjects had only two normal alleles and no expanded allele. Revision of the clinical data casts doubt on the original diagnosis of DM in their families. Preferential amplification of the normal non-expanded allele was noted in three asymptomatic gene carriers in this study (as well as in two of their clinically affected relatives). We caution that, at least in our hands, the DM mutation can be confidently excluded by this PCR based method only if both normal alleles have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)


Specific molecular prenatal diagnosis for the CTG mutation in myotonic dystrophy. The results of DNA analysis for the specific mutation of myotonic dystrophy are reported in eight pregnancies (two studied retrospectively) in six families. Four results were normal; in the other four, large DNA expansions were found, comparable to the range seen in severely affected children with congenital onset of the disorder. The results agreed with those obtained by linked DNA markers in the six cases where they were available. We conclude that specific molecular prenatal diagnosis of myotonic dystrophy is feasible, and that an abnormal result may also give a guide to possible severity, though this should be interpreted with caution until greater experience is available.


Expansion of the myotonic dystrophy gene in Italian and Spanish patients. Myotonic dystrophy results from expansion of a (CTG)n repeat at the 3' untranslated region of the myotonin-protein kinase gene. We show here the genomic analysis of 322 symptomatic patients with the cDNA-25 probe detecting disease specific EcoRI restriction fragments. The expansion was found in the majority of Italian and Spanish patients (92%). The implications of these results for the detection of symptomatic patients in southern Europe are discussed.


Intestinal pseudo-obstruction in myotonic dystrophy. We describe four myotonic dystrophy (DM) patients who developed recurrent intestinal pseudo-obstruction. Some episodes were associated with gastroenteritis, while abdominal crowding may have occurred in one case during the third trimester of pregnancy. In most instances, however, no apparent cause could be identified. Intestinal pseudo-obstruction may occur at any stage of DM. In one of our cases intestinal pseudo-obstruction preceded significant muscle weakness by 15 years. Intestinal pseudo-obstruction is usually treated effectively with conservative measures. These include restriction of oral intake, intravenous fluids, and multiple enemas or colonoscopy. Improved intestinal function was noted in one case treated with the prokinetic agent cisapride. A partial sigmoid resection was performed in three cases with dolichomegacolon. No abnormalities were reported on histological examination. Since intestinal pseudo-obstruction is a rare complication of DM, it is of interest that two of our cases are sibs. Review of published reports showed several reports of familial occurrence of specific complications. These include cardiac conduction disturbances, focal myocarditis, mitral valve prolapse, pilomatrixomas, polyneuropathy, normal pressure hydrocephalus, and dilatation of the urinary tract. Myotonic dystrophy may show a tendency to familial clustering of organ specific involvement.


Inheritance of the fragile X syndrome: size of the fragile X premutation is a major determinant of the transition to full mutation. The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat. Two main types of mutation have been categorised. Clinical expression is associated with the presence of the full mutation, while subjects who carry only a premutation do not have mental retardation. Premutations have a high risk of transition to full mutation when transmitted by a female. We have used direct detection of the mutations to characterise large families who illustrate the wide variation in penetrance which has been observed in different sibships (a feature often called the Sherman paradox). A family originally found to show tight genetic linkage between the factor 9 gene and the fragile X locus was reanalysed, confirming the original genotype assignments and the observed linkage. The size of premutations was measured by Southern blotting and by using a PCR based test in 102 carrier mothers and this was correlated with the type of mutation found in their offspring. The risk of transition to full mutation was found to be very low for premutations with a size increase (delta) of about 100 bp, increasing up to 100% when the size of premutation was larger than about 200 bp, even after taking into account (at least partially) ascertainment bias. These results confirm and extend those reported by Fu et al (1991) and Yu et al (1992) and explain the Sherman paradox.(ABSTRACT TRUNCATED AT 250 WORDS)


Frequent small amplifications in the FMR-1 gene in fra(X) families: limits to the diagnosis of 'premutations'. In five of 40 fra(X) families reinvestigated using the new intragenic probe StB12.3, small amplifications of the DNA fragment appeared unexpectedly in addition to the mutations found in the probands. This suggests that enlargements of the FMR-1 gene detectable by Southern blotting using this probe must be present at an appreciable frequency in the general population. A proportion of these may be classifiable as 'premutations', or precursors of the much amplified, hypermethylated, and somatically unstable fragment associated with the fragile X syndrome, while others will merely represent stable polymorphisms in fragment length. Hence, accurate diagnosis of some fra(X) carriers will depend upon a more precise measurement of insert size than is currently provided by the newly available molecular probes.


Duplication within chromosome 17p11.2 in 12 families of French ancestry with Charcot-Marie-Tooth disease type 1a. The French CMT Research Group. Hereditary motor and sensory neuropathy type I (HMSN I), also designated Charcot-Marie-Tooth disease type 1 (CMT1), is a peripheral neuropathy frequently inherited as an autosomal dominant trait, characterised by progressive distal muscular atrophy and sensory loss with markedly decreased nerve conduction velocity. A duplication within chromosome 17p11.2, cosegregating with the disease, has recently been reported in several CMT1a families. In order to estimate the frequency of this anomaly and determine the location of a duplication in this region, 12 CMT1 families were analysed with polymorphic DNA markers located within 17p11.2-12. Duplications were found in all families including loci D17S61 (EW401), D17S122 (VAW409R3a and RM11-GT), and D17S125 (VAW412R3). The duplications were completely linked and associated with the disease (lod score of 20.77 at zero recombination). Screening for the RM11-GT microsatellite showed that most of the duplicated haplotypes were heterozygous, supporting the hypothesis that the duplication resulted from an unequal crossing over. There was no significant haplotype association within the duplicated region suggesting that the duplication resulted de novo as an independent event in each family. In one family, recombination within the duplicated region was observed, indicating that genetic instability in 17p11.2 might be related to a high recombination rate. Since most cases of CMT1a seem to result from this segmental trisomy, it can be used as a basis for DNA diagnosis of the disease.


High frequency of the Lebanese allele of the LDLr gene among Brazilian patients with familial hypercholesterolaemia. We analysed the LDL receptor (LDLr) gene in 18 Brazilian patients with familial hypercholesterolaemia (FH) from 10 unrelated families. The combination of a direct search for the Lebanese allele of the LDLr gene by a PCR method and Southern blotting using cDNA probes allowed the identification of the gene defect in six out of 10 families. The Lebanese allele was found in five families and in one family the disease was caused by a 4 kb deletion in the 3' half of the LDLr gene. The results indicate an important contribution of the Lebanese allele to the prevalence of FH in the Brazilian population and suggest that it may also be the most common cause of FH in other mixed populations outside the Middle East.


Clinical reinvestigation and linkage analysis in the family with Episkopi blindness (Norrie disease). We present the results of a clinical and genetic reinvestigation of the Cypriot family affected by an X chromosomally inherited eye disease originally published by Taylor et al, who coined the term Episkopi blindness. The pedigree was extended to 160 members, including 16 affected males out of 48 males at risk for the disease, most of whom were seen by one of us (PA). Affected males are blind with no associated symptoms and apparently are not mentally retarded. Thirty-nine family members agreed to blood sampling for genetic investigations. RFLP analysis was performed using probes from the region known to be deleted in some Norrie patients and polymorphic markers (DXS77, DXS7, MAOA, DXS255) from the proximal short arm of the X chromosome. There was no deletion for any of the probes in the affected males. Linkage analysis yielded positive lod scores for all informative markers (Z (DXS255, theta = 0) = 6.54, Z (MAOA, theta = 0) = 2.23, Z (DXS7, theta = 0) = 2.13). Thus, the conclusion that Episkopi blindness and Norrie disease (NDP, MIM *310600) are the same entity based on clinical evidence is now reinforced by gene mapping.


Factors affecting the uptake of prenatal diagnosis for sickle cell disease. Between 1979 and 1990, 170 couples at risk of having children with sickle cell disease, resident in the UK and with a continuing pregnancy, were referred for counselling at the University College Hospital Perinatal Centre. Approximately 50% of the couples, including those where one partner actually had sickle cell disease, requested prenatal diagnosis. This was requested in 82% of pregnancies when the mother was seen in the first trimester of pregnancy and in 49% when she was seen in the second trimester. More than 90% of referred couples who already had an affected child requested prenatal diagnosis. The type of sickle cell disease involved and ethnic group also influenced choice. These results show the importance of detecting and counselling couples at risk before pregnancy whenever possible.


Polyglandular autoimmune syndrome type I among Iranian Jews. Polyglandular autoimmune syndrome (PAS) has been well characterised and the accepted criteria for diagnosis are the presence of at least two of the three major components: hypoparathyroidism (HPT), candidiasis, and adrenal insufficiency (AI). HPT may, however, be the only manifestation of the syndrome. Iranian Jews, having a high rate of consanguinity, appear to be a community in which PAS type I is frequent. We report on 19 families of patients with HPT from the Iranian Jewish community assuming that they are in fact affected with PAS type I. In the 19 families, 23 patients were affected, including 11 males and 12 females. All the patients but one had HPT (96%), and most were diagnosed by the age of 20 years (91%). AI was diagnosed in five of our patients; in all cases but one it appeared after HPT. Mild oral candidiasis was present in four patients and six of the patients (three males and three females) had hypogonadism. Other features of the syndrome found in some of our patients were pernicious anaemia, hypothyroidism, and alopecia. The disease is autosomal recessive and the calculated prevalence among the Iranian Jews is 1:6500 to 1:9000. The disease is also found with a very high incidence among Finns. A comparison of the symptoms between the two groups showed clinical differences including the relative rarity of candidiasis and absence of keratopathy among the Iranian Jews.


Orofaciodigital syndrome type I in a girl with unilateral tibial pseudarthrosis. The orofaciodigital syndromes are a group of possibly seven different malformation syndromes including oral, facial, and digital malformations. Type I has X linked dominant inheritance whereas the other types show autosomal recessive inheritance. An exact diagnosis is therefore important for genetic counselling. We here report a girl with orofaciodigital syndrome type I. She had cystic kidney disease at the age of 8 months which has not previously been reported in an infant with orofaciodigital syndrome. In addition she had unilateral tibial pseudarthrosis which has only rarely been reported in the orofaciodigital syndromes and in type II only.


Lethal skeletal dysplasia owing to double heterozygosity for achondroplasia and spondyloepiphyseal dysplasia congenita. A male infant with lethal short limbed dwarfism is described. His father had spondyloepiphyseal dysplasia congenita and his mother had achondroplasia. It is believed that the infant inherited both of these disorders and that their combined effects resulted in early death owing primarily to severe pulmonary hypoplasia.


An atypical form of mucolipidosis III.

An atypical form of mucolipidosis III. We report two sibs showing a very mild form of mucolipidosis III with no clinical signs but isolated involvement of the hip and very mild abnormalities of the spine. This indicates that a storage disease, in particular mucolipidosis III, should be considered in any case of isolated bilateral hip dysplasia. The differences from other reported atypical variants of mucolipidosis III are discussed.


Numerous group I introns with variable distributions in the ribosomal DNA of a lichen fungus. The length of the small subunit ribosomal DNA (SSU rDNA) differs significantly among individuals from natural populations of the ascomycetous lichen complex Cladonia chlorophaea. The sequence of the 3' region of the SSU rDNA from two individuals, chosen to represent the shortest and longest sequences, revealed multiple insertions within a region that otherwise aligned with a 520-nucleotide sequence of the SSU rDNA in Saccharomyces cerevisiae. The high degree of variability in SSU rDNA size can be accounted for by different numbers of insertions; one individual had two group I introns and the second had five introns, two of which were clearly related to introns at identical positions in the other individual. Yet, introns in different positions, whether within an individual or between individuals, were not similar in sequence. The distribution of introns at three of the positions is consistent with either intron loss or acquisition, and clearly indicates the dynamic variability in this region of the nuclear genome. All seven insertions, which ranged in size from 210 to 228 nucleotides, had the conserved sequence and secondary structural elements of group I introns. The variation in distribution and sequence of group I introns within a short highly conserved region of rDNA presents a unique opportunity for examining the molecular evolution and mobility of group I introns within a systematics framework.


An accurate method for determining the helical repeat of DNA in solution reveals differences to the crystal structures of two B-DNA decamers. Many DNA sequences have been studied by X-ray crystallography with the goal of deciphering a sequence-structure code. We have determined the helical repeats of two B-type DNA decamers in solution employing an electrophoretic method based on phasing of bent segments. The decamers contain recognition sites for the dcm methyltransferase and for the restriction nuclease NarI with a mutational hotspot. Their helical repeats are 10.59(+/- 0.05) bp and 10.52(+/- 0.03) bp, respectively, whereas crystallographic analysis yielded 10.0 bp in the solid state. This difference is greater than that for the transition between B- and A-type DNA in solution. Thus, reliable information about the polymorphism of DNA in solution must be based on both X-ray and solution data. We describe a generally applicable approach to accurately determine helical repeats of small DNA duplexes in solution.


Chromatin reconstitution on small DNA rings. V. DNA thermal flexibility of single nucleosomes. The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles. The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted. The experiment was as follows. The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded. Second, the gel temperature was increased so that the former mobility was restored. Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition. The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively. The figure, 0.69 (+/- 0.07), was used to derive the length of DNA in interaction with the histones, 109 (+/- 25) bp. Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy. This consistency further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as "single-turn" particles. The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the "single-turn" particle notion may be extended to this particular case of naturally-occurring H1-free chromatin. However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes. It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.


A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease. Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.


Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue. Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.


Absence of the thermal transition in apo-alpha-lactalbumin in the molten globule state. A study by differential scanning microcalorimetry. To estimate the energy level of the molten globule state, the heat capacity function of apo-alpha-lactalbumin in the molten globule state has been examined using a scanning microcalorimeter at neutral pH. The results showed that the enthalpy difference between the molten globule state and presumed unfolded state by heating was almost zero at neutral pH, demonstrating that the molten globule state does not exhibit any co-operative transition upon heating. This is in agreement with the results already reported at acid pH, but is apparently in conflict with that recently reported with some assumptions at neutral pH.


Folding on the ribosome of Escherichia coli tryptophan synthase beta subunit nascent chains probed with a conformation-dependent monoclonal antibody. Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.


General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase. A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.


Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20. The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.


A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III. We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.


In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage. Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (IndS) and here called IndS, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these IndS mutant proteins, and of several double mutant proteins containing an IndS mutation and one of several mutations, termed Ind-, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the IndS mutant proteins had a higher maximum rate and a lower apparent pKa than wild-type LexA. Second, the IndS mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of Kcat/Km was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind- mutant protein. We interpret these data to mean that the IndS mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.


Early stages in RecA protein-catalyzed pairing. Analysis of coaggregate formation and non-homologous DNA contacts. RecA protein will catalyze the in vitro pairing of homologous DNA molecules. To further explore the events involved in the search for homology, we have applied a nitrocellulose filter binding assay to follow pairing, and a sedimentation assay to follow the generation of aggregates (termed coaggregates) formed between RecA-complexed single-stranded (ss) DNA and double stranded (ds) DNA. Electron microscopy (EM) was used to visualize the structures involved. RecA protein promoted the pairing of circular M13 ssDNA and linear M13mp7 dsDNA efficiently in the absence of coaggregates. Indeed, pairing of homologous ss- and dsDNAs involved coaggregate formation only if the dsDNA was circular. For DNAs containing only a few hundred base-pairs of homology, for example pUC7 dsDNA and M13mp7 ssDNA, pairing and joint formation was observed if the dsDNA was superhelical but not if it was topologically relaxed or linear with the homology internal to an end of the dsDNA. The effect of non-covalently attached heterologous dsDNA on the RecA-promoted joining of M13 ssDNA and linear M13mp7 dsDNA (with non-M13 sequences at both ends) was found to depend on the topology and concentration of the heterologous DNA. A tenfold excess of superhelical pBR322 DNA strongly inhibited pairing. However, addition of relaxed or linear pBR322 DNA to the pairing reaction had little effect. As seen by EM, superhelical pBR322 DNA inhibited joint formation by excluding the homologous dsDNA form the coaggregates. EM also revealed heterologous DNA interactions presumably involved in the search for homology. Here the use of EM has provided a direct visualization of the form and architecture of coaggregates revealing a dense interweaving of presynaptic filaments and dsDNA.


Structure of the pericentric long arm region of the human Y chromosome. We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome. The structures of one cosmid and three yeast artificial chromosome clones were determined. The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families. A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.


DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing. The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.


Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain. Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.


Two tRNA-binding sites in addition to A and P sites on eukaryotic ribosomes. The interaction of tRNA with 80 S ribosomes from rabbit liver was studied using biochemical as well as fluorescence techniques. Besides the canonical A and P sites, two additional sites were found which specifically bind deacylated tRNA. One of the sites is analogous to the E site of prokaryotic ribosomes, in that binding of tRNA is labile, does not depend on codon-anticodon interaction, does not protect the anticodon loop from solvent access, and requires the presence of the 3'-terminal adenosine of the tRNA. In contrast, the stability of the tRNA complex with the second site (S site) is high. tRNA binding to the S site is also codon-independent; nevertheless, the anticodon loop is shielded from solvent access. Removal of the 3'-terminal adenosine decreases the affinity of tRNA(Phe) for the S site approximately 50-fold. tRNA(Phe) is retained at the S site during translocation and through poly(Phe) synthesis. Thus, the S site does not seem to be an intermediate site for the tRNA during the elongation cycle. Rather, the tRNA bound to the S site may allosterically modulate the function of the ribosome.


Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides. The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.