Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Climate deteriorations and Neanderthal demise in interior Iberia.

Time and circumstances for the disappearance of Neanderthals and its relationship with the advent of Modern Humans are not yet sufficiently resolved, especially in case of the Iberian Peninsula. Reconstructing palaeoenvironmental conditions during the last glacial period is crucial to clarifying whether climate deteriorations or competition and contacts with Modern Humans played the pivotal role in driving Neanderthals to extinction. A high-resolution loess record from the Upper Tagus Basin in central Spain demonstrates that the Neanderthal abandonment of inner Iberian territories 42 kyr ago coincided with the evolvement of hostile environmental conditions, while archaeological evidence testifies that this desertion took place regardless of modern humans' activities. According to stratigraphic findings and stable isotope analyses, this period corresponded to the driest environmental conditions of the last glacial apart from an even drier period linked to Heinrich Stadial 3. Our results show that during Marine Isotope Stages (MIS) 4 and 2 climate deteriorations in interior Iberia temporally coincided with northern hemisphere cold periods (Heinrich stadials). Solely during the middle MIS 3, in a period surrounding 42 kyr ago, this relation seems not straightforward, which may demonstrate the complexity of terrestrial climate conditions during glacial periods.

Identification of cytochrome P-450, and its distribution in the membrana granulosa of the preovulatory follicle, using quantitative cytochemistry.

The distribution of cytochrome P-450 (C-P450), an essential component of the oxidative enzyme system involved in the hydroxylation of steroids, was measured by quantitative cytochemistry in cryostat sections of preovulatory follicles obtained from rats in proestrous. The sections were reacted with medium saturated with carbon monoxide with or without the addition of sodium dithionite. The absorbance spectrum was measured from 400 to 500 nm and a difference spectrum calculated by subtracting the extinction obtained from incubations without sodium dithionite from that obtained in the presence of sodium dithionite. A distinct peak at 450nm was recorded in cells of the peripheral portion of the membrana granulosa (MG) but not in those of the cumulus, providing evidence for the presence and differential distribution of C-P450 in the MG of the preovulatory follicle.

Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis.

The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Hantavirus strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nephropathia epidemica virus strain Hällnäs B1 (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level.

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Evidence that Borrelia burgdorferi immunodominant proteins p100, p94 and p83 are identical.

Recently there have been reports on high-molecular mass components of Borrelia burgdorferi, namely the p100, p94 and p83, which claimed these proteins to be specific marker antigens for the serodiagnosis of late Lyme borreliosis. The nucleotide sequences of the p100 and p83 have been published. The alignment of the deduced N-terminal amino acid sequences with the N-terminal sequence of the p94 now provides evidence that all three proteins are identical.

Effects of Silastic progesterone implants on activity cycles and steroid levels in ovariectomized and intact female rats.

We have previously shown that although Silastic implants of progesterone reduce the amount of running of animals living in activity wheels, progesterone-treated animals continue to show periodic fluctuations or peaks in activity. We hypothesized that although progesterone treatment inhibited estrous cycles, ovaries of animals treated with Silastic implants of progesterone continued to secrete estradiol in amounts adequate to stimulate moderate levels of running. In the present study we tested this hypothesis by removing ovaries from progesterone-treated animals and comparing their running behavior and steroid levels to progesterone-treated animals who received sham ovariectomies. Although progesterone treatment significantly inhibited running activity, removal of ovaries in progesterone-treated animals further suppressed running activity. In addition, both estradiol and progesterone levels were significantly reduced following removal of ovaries in progesterone-treated animals. We conclude that although Silastic progesterone implants inhibit normal ovarian and estrous activity cycles, ovaries produce sufficient estradiol to stimulate running behavior.

Severe hemorrhagic complications from infection with nephropathia epidemica strain of Hantavirus.

In the following we describe a case of severe hemorrhagic fever with renal syndrome (HFRS) caused by Puumala infection. The diagnosis was made by immunofluorescence technique and by solid phase enzyme immunoassay using recombinant nucleocapsid antigen of a Puumala serotype strain. Such a clinical course with severe bleeding complications is considered untypical for Puumala induced HFRS.

Spontaneous malignomas in Tupaia (tree shrew).

Nine spontaneous malignomas of the tree shrew were detected and analysed during an observation period of nine years. The tumours were histopathologically examined and classified. All malignomas developed in imported Tupaia only. From the tumour cells of two different animals new Tupaia herpesviruses were isolated. This is the first. report on spontaneous malignomas of Tupaia in captivity.

High prevalence of genetically diverse Borrelia bavariensis-like strains in Ixodes persulcatus from Selenge Aimag, Mongolia.

In Mongolia, Lyme borreliosis was first reported in 2003. To determine which Borrelia species may contribute to the occurrence of Lyme borreliosis in Mongolia, real-time PCR was conducted on 372 adult Ixodes persulcatus ticks collected in Selenge Aimag, the province with the highest incidence of human Lyme borreliosis. 24.5% of ticks were identified to be positive for Borrelia burgdorferi sensu lato DNA. Species differentiation using an SNP-based real-time PCR and multi-locus sequence analysis revealed that strains phylogenetically closely related to B. bavariensis (previously known as B. garinii OspA serotype 4) is the most prevalent species, showing an unexpectedly high genetic diversity.

An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis.

In the present article, the detection and the development of a parasitic endocytobiont within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis is presented. An otherwise healthy 55-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of a corneal trauma. Acanthamoebae as well as other smaller free-living amoebae could be detected from the fluid of the contact lens storage cases by culture methods. A successful therapy could be provided consequently. Two of these Acanthamoeba strains showed intracellular aggregating organisms. Within 2 to 3 days, the host amoebae ruptured, and numerous microorganisms were released. We succeeded in detecting the mechanism of infection and intrusion of this organisms by using light and electron microscopy. Infection with this endocytobiont is a suitable model for studying the host-parasite relations while the parasites use their hosts as so-called Trojan horses (see Barker, Lambert, Brown, Infect Immun 61:3503-3510, 1992).

Telemicrobiology: a novel telemedical module for mission support in the field of infectious medicine.

Infectious diseases are among the most common diseases suffered by soldiers while serving in missions away from their home countries. The diagnosis of theses diseases requires special procedures and expertise, both of which are provided by field microbiological laboratories. In order to support the diagnostic process by means of telemedicine, a modification of the standard telemedicine workstation, i.e. a telemicrobiology module with special equipment, camera and software, has been designed and validated. This module, currently in use in two operational theaters, has stood the test in routine practice. It enables the transmission of high-quality static images of microscopic specimens or overgrown nutrient media in a matter of seconds. The inclusion of experts into diagnostic analysis through the use of telemedicine improves diagnostic specificity by avoiding false positive results and, particularly in medical parasitology, allows a treatment-essential diagnosis without the dispatch of specimens to Germany. Telemicrobiology allows the control of the entire microbiology diagnostic process by expert workstation even with only a microbiological technician on site.

Effects of tetrahydrocannabinol on rat preovulatory follicles: a quantitative cytochemical analysis.

Using a microdensitometric histochemical assay, delta 5-3 beta-hydroxysteroid dehydrogenase activity and glucose-6-phosphate dehydrogenase Types I and II hydrogen generation were measured in preovulatory follicles from normal rats, and in follicles from rats given tetrahydrocannabinol for three days prior to sacrifice. Hydroxysteroid dehydrogenase and Type I hydrogen generation are involved in steroidogenesis, whereas Type II hydrogen generation is involved with general cellular metabolism. All ovaries were removed on pro-oestrus, frozen, sectioned and the sections reacted with the appropriate media. Enzyme activity was measured in the theca and in three regions of the membrana granulosa; peripheral, antral and corona radiata. Compared to control animals, the hydroxysteroid dehydrogenase activity was significantly reduced in all follicular regions in rats exposed to tetrahydrocannabinol. Type I hydrogen generation was significantly less in the theca and peripheral region of preovulatory follicles from rats given tetrahydrocannabinol, but the same in the antral region and corona radiata. In all follicular regions examined, Type II hydrogen generation was unchanged following tetrahydrocannabinol administration. Thus, only the enzymes specifically associated with follicular steroidogenesis were affected by administration of the drug.

A comparison of rat and hamster preovulatory follicles: an examination of differences in morphology and enzyme activity using qualitative and quantitative analyses.

The structure and enzymatic content of rat and hamster preovulatory follicles were examined using morphologic, qualitative, and quantitative cytochemical techniques. Interfollicular structure in both types was similar but lipid droplet distribution differed. The rat theca contained many more droplets than the hamster, while the hamster membrana granulosa contained more droplets than the rat. delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) activity and glucose-6-phosphate dehydrogenase (G-6-PD) Types IH and IIH generation were measured using quantitative cytochemistry. Rat theca and granulosa contained more 3 beta OHD activity than found in comparable regions in hamster. G-6-PD Type IH and Type IIH generation were the same in comparable regions of rat and hamster, but in rat there was more Type IIH than IH, while the converse existed in hamster. These differences and their relationship to follicular steroid production are discussed.

A quantitative electron microscopic analysis of the membrana granulosa of rat preovulatory follicles.

The ultrastructure of the membrana granulosa (MG) of rat preovulatory follicles was examined using stereological techniques. Organelles studied were nuclei, mitochondria, lipid droplets (LD), lysosomes, and smooth and rough endoplasmic reticulum (SER, RER). The peripheral region of the MG contained the greatest volume of mitochondria, LD and SER, organelles associated with steroidogenesis. The volume of RER, an organelle associated with protein production, was greatest in the cumulus oophorus. These results corroborate previous analyses and demonstrate that the rat MG is composed of discrete subregions.

Western blot as a tool in the diagnosis of Lyme borreliosis.

Borrelia burgdorferi is the causative agent of Lyme borreliosis, a multisystem disorder, which can mimic numerous immune disorders and inflammatory diseases. Laboratory diagnosis of Borrelia infection relies on immunodiagnostic assays, which, however, are hampered by unsatisfactory specificity. The Western blot technique has been employed to analyze the humoral immune response in Lyme borreliosis and is used as a serodiagnostic confirmation test. The most important immunodominant proteins of Borrelia burgdorferi are the 94 kDa, 60 kDa, 41 kDa (flagellin), 34 kDa (Osp B), 31 kDa (Osp A), 30 kDa, 21 kDa (Osp C), and 17/18 kDa proteins. Whereas the 60 kDa, 41 kDa, and 34 kDa constituents reveal a marked cross-antigenicity with other spirochetes and even more distantly related bacteria, antibodies against the 94 kDa, 31 kDa and 21 kDa proteins are largely species-specific. The early immune response in Lyme borreliosis is triggered mainly by the flagellin. In the later stage a wide range of immunogenic proteins is involved, with the 94 kDa antigen being the best marker for late immune response. If the Western blot is used for diagnostic purposes the differences between early and late-stage immunogenicity of Borrelia proteins must be taken into account. Interpretation criteria for blot positivity in early-stage borreliosis are primarily based on the presence of the 21 kDa band and the semiquantitatively recorded intensity of the 41 kDa band. In the diagnosis of late-stage infection, blot positivity relies on the presence of the 94 kDa, 39 kDa, 31 kDa, 30 kDa and 21 kDa bands.

Humoral immune response against Helicobacter pylori as determined by immunoblot.

An immunoblot method has been evaluated to diagnose Helicobacter pylori infection serologically by comparing 69 serum specimens from patients with a positive Gram stain and/or culture result and a positive urease test on biopsy material, as well as 51 serum specimens from patients with at least 4 negative urease tests, and negative microscopy and culture results. Sensitivity and specificity was found to be 100%. Recognition of the cross-reacting flagellin (66 kDa), flagellar sheath protein (51 kDa), and a 14 kDa protein are not a criterion for a current H. pylori infection. On the other hand, any combination of at least two of the 180, 120, 90, 75, 67, 29.5 and 19 kDa bands were diagnostic of infection. Three H. pylori strains, which were compared with both gel electrophoretic analyses and immunoblot reactivity, exhibited in part strong qualitative and quantitative differences that particularly affect the 120 kDa pathogenic factor, the large urease subunit and other proteins especially in the molecular mass range from 50 to 67 kDa. IgG immunoblot patterns showed that the choice of H. pylori strain, as well as a reproducible and standardizable antigen preparation, is of great importance for the reliability of serodiagnostic tests. The immunoblot method was found to be a valuable tool for the semi-quantitative confirmation of results achieved with other serological methods as well as optimization and quality control of the antigens used for serodiagnostic purposes.