Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.     

CD97, but not its closely related EGF-TM7 family member EMR2, is expressed on gastric,pancreatic, and esophageal carcinomas

CD97 expression is related closely to the dedifferentiation and tumor stage in thyroid carcinomas. We systematically examined the role of CD97 and its closest relative, EMR2, in normal and malignant gastric, esophageal, and pancreatic tissue. The normal tissues were EMR2-, whereas CD97 was expressed slightly in the parietal cells of gastric mucosa and in exocrine pancreatic cells. Interestingly, intralobular and interlobular pancreatic ducts were CD97+. All tumors were EMR2-. CD97 was expressed by 44 of 50 gastric, 14 of 18 pancreatic, and 10 of 13 esophageal carcinomas. Of the 44 gastric cancers, 27 showed disseminated or scattered tumor cells at the invasion front with stronger CD97 expression than tumor cells located in solid tumor formations. There was no correlation between CD97 levels in the tumors or soluble CD97 in the serum samples and the clinicopathologic features of the patients. Taken together, significant numbers of gastric, esophageal, and pancreatic carcinomas are CD97+, whereas its homolog, EMR2, does not have any role in such tumors.     

Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana

All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV.     

The effect of stimulus velocity on the response of movement sensitive neurons of the frog's retina

Summary 1. By means of metal-filled micropipettes the action potentials of 4 different classes of optic nerve fibers were recorded in Rana esculenta. The relationship between the angular velocity of the stimuli and the neuronal response was determined.2. If an object smaller than the excitatory receptive field (ERF) was moved through the receptive field of the different classes of retinal units the response depended on the angular velocity, contrast and size of the stimulus. The response was measured as the average impulses frequency (R) during the traverse of the ERF. Between R and the angular velocity (v) the equation R=k·v ^c [impulses · sec^–1] was found. The exponent c was 0.5 for class 1 neurons, 0.7 for class 2 neurons, and 0.95 for class 3 neurons. In class 4 neurons the response to large stimuli increased linearly with the increase of the angular velocity, while no systematic relationship between R and v was valid for small moving stimuli (<5°)3. If the contrast or the size of the stimuli was changed the exponent c was not changed; but k depended on both parameters and on the direction of the contrast against the background. The power function was no longer valid if stimuli considerably larger than the ERF were used. The exponent c was independent of the type of the movement (linear, non-linear, irregular movement); it was also independent of the direction of the motion.4. A model of the receptive field is demonstrated. In this model an RC-filter function within the bipolar cells is assumed. The bipolar cells with different filter function activate different classes of ganglion cells. Different time constants of the bandpass filter at the bipolar cell level are the main cause for the different exponents of the power function between angular velocity and neuronal response.     

Kollagenfolien als temporärer Hautersatz. Klinische und patho-anatomische Untersuchungen

Zusammenfassung Im Kaninchenversuch wurde eine neue Kollagenfolie (COL 65–70, 65–71) im Vergleich mit Polypyrrolidonpulver, Teflonfilz und unbehandelten Kontrollen zur temporären Hautdeckung nach flächenhafter Hautverbrennung getestet. Bei der verwandten Kollagenfolie handelt es sich um gereinigtes tierisches Kollagen, das nach seiner Lösung wieder gefällt wurde. Nach einem patentierten Verfahren wurden hierbei die Moleküle wieder ausgerichtet, so daß eine gewisse physikalische Festigkeit gewährleistet ist. Ein Weichmacherzusatz bewirkt die Geschmeidigkeit. Zur Verhinderung der unerwünschten Austrocknung nach Auflage auf die Wundfläche wurde die Kollagen-Gelplatte mit einer härteren Kollagenfolie abgedeckt. Eine feingeweblich erfaßbare Abwehrreaktion des Wirtsorganismus gegen die Kollagenfolie bzw. eine entzündliche Demarkation konnte an unserem Versuchskollektiv nicht nachgewiesen werden.

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Summary Along with polypyrrolidon powder and teflon felt, a new collagen foil has been evaluated for the temporary closure of skin defects caused by experimental third degree burns in rabbits. A control serics remained untreated. The collagen foil applied was obtained from animal connective tissues the fibrils of which were regenerated after liquefaction. According to a patent procedure the collagen molecules were reconverted. By the addition of a weakener the flexibility of the foil could be changed. In order to avoid an undesired exsiccation after covering the burn wound the collagen foil was protected by a tanned collagen film layer. In the present material, no defensive reactions of the recipient organisms against the collagen foil nor any inflammatory demarcations could be noticed by means of microscopic examination.

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Herrn Prof. Dr. Drs. h. c. Dr. h. c.K. H. Bauer zum 75. Geburtstag in Verehrung und Dankbarkeit zugeeignet.     

Antibiotic sensitivities of urinary pathogens, 1971-8.

The sensitivites of urinary pathogens from general practice and from hospital to a range of antimicrobial drugs have been recorded for the period 1971-8. There have been changes in the proportions of the different bacterial species and in their sensitivites to antibiotics. In particular, the position of ampicillin/amoxycillin and cephalosporins has deteriorated, partly because more resistant species have somewhat increased in prevalence and partly because the usually sensitive species, such as Escherichia coli, have become more resistant. Over the period 1971-8 the sensitivity of urinary pathogens, whether in general practice or in hospital, to co-trimoxazole and to trimethoprim has been maintained at a high level.     

Clone-based systematic haplotyping (CSH): a procedure for physical haplotyping of whole genomes

We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing approximately 10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual.     

Voluntary HIV counseling and testing: experience among the sexually active population in Bangui,Central African Republic

OBJECTIVES: In July 1997, the National Reference Center for Sexually Transmitted Diseases of Bangui, Central African Republic (CAR), was expanded by the creation of an anonymous and voluntary counseling and testing (VCT) unit for HIV infection, the Anonymous Surveillance Unit (Unité de Dépistage Anonyme [UDA]). The goal of the UDA was to initiate and promote voluntary HIV testing in the general adult population of Bangui. We carried out an observational and comprehensive survey over a 4-year period to document and analyze the experience of VCT in the UDA, with special attention to risk factors associated with HIV infection. METHODS: All clients for VCT were given adequate pretest counseling by trained counselors focused on knowledge about HIV infection and sexually transmitted infections, individual risks of acquiring HIV, and anticipation of the client's attitude about test results. After consent was obtained, a blood sample was drawn and tested for HIV by two ELISAs in parallel. The client paid a standard cost of $1.20 at the initial visit. After a week, test results were given to the client during the posttest visit, at which time HIV-seropositive individuals received emotional support and were referred to specific social or medical structures. Seronegative clients received reinforcement of prevention messages and were asked to come back for serologic follow-up free of charge after 3 (M3) and 12 (M12) months. RESULTS: From July 1997 to March 2001, 5686 individuals aged 14 to 65 years (mean age, 27 years) had an initial visit for VCT (V1). Peaks of UDA visitation (250-450 clients) were observed on the annual AIDS Day in the CAR, at which time HIV serologic testing was offered free of charge. A total of 5060 (89%) clients came back for a second visit (V2) to receive test results. Among those, 18.3% were infected with HIV type 1. Multivariate analysis of risk factors demonstrated marked association of HIV seropositivity with age, female gender, widowed/divorced women, poor or low education level, occupations such as civil servants or merchants, presence of symptoms of sexually transmitted infections, and lack of systematic condom use. Single young women were at higher risk for HIV infection compared with men of the same age (OR = 7.7 for women aged 15-24 years, 95% CI: 4.0-14.0; OR = 2.8 for women aged 25-34 years, 95% CI: 1.7-4.5). Widowed women older than 44 years of age were more likely to be HIV-seropositive than men (OR = 10.0; 95% CI: 1.7-83.6). A total of 885 (21%) HIV-seronegative individuals returned for follow-up at 3 months (M3; 0.45% rate of seroconversion). Seventy-nine (9%) individuals returned at 12 months (M12), without any new cases of HIV infection. HIV-negative clients consulting at M3 and M12 showed a significant reduction in unprotected intercourse with occasional sexual partners. CONCLUSION: This experience demonstrates that VCT for HIV infection is feasible in Central Africa.     

Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody.Using this technique we have been able to identify the precipitate arc in crossed immunoelectrophoresis of major histocompatibility complex (MHC) class I molecules in a mixture of all detergent solubilized cell membrane molecules by means of a monoclonal antibody, the specificity of which was known independently to be against MHC class I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains.