Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway.

Microtubules of all eukaryotic cells are formed by α- and ß-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and ß-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

Microtubule drugs: action, selectivity, and resistance across the kingdoms of life.

Microtubule drugs such as paclitaxel, colchicine, vinblastine, trifluralin, or oryzalin form a chemically diverse group that has been reinforced by a large number of novel compounds over time. They all share the ability to change microtubule properties. The profound effects of disrupted microtubule systems on cell physiology can be used in research as well as anticancer treatment and agricultural weed control. The activity of microtubule drugs generally depends on their binding to α- and ß-tubulin subunits. The microtubule drugs are often effective only in certain taxonomic groups, while other organisms remain resistant. Available information on the molecular basis of this selectivity is summarized. In addition to reviewing published data, we performed sequence data mining, searching for kingdom-specific signatures in plant, animal, fungal, and protozoan tubulin sequences. Our findings clearly correlate with known microtubule drug resistance determinants and add more amino acid positions with a putative effect on drug-tubulin interaction. The issue of microtubule network properties in plant cells producing microtubule drugs is also addressed.

[Invasive streptococcal infections]. / Invazivní streptokokové infekce.

The severity of streptococcal infections depends upon different virulence of individual strains of its causative agent. The most important species are beta-haemolytic group A streptococci (GAS). Clinical manifestations include skin affections, respiratory tract infections and, in particular, serious systemic invasive infections. The pathogenicity of GAS is derived from cell wall components and extracellular products, especially toxins with properties of the so-called superantigens. Less invasive forms of the disease are include necrotizing fasciitis, myositis, pneumonia, sepsis without focus, arthritis, meningitis, puerperal sepsis, streptococcal toxic shock syndrome (STSS) and severe course of erysipelas and cellulitis with blood culture positive for GAS. In most cases, soft tissue infections dominate, often accompanied by chronic diseases of lower extremities in elderly patients. The other clinical forms are rather rare. In children, the condition is clearly frequently related to chickenpox. The generally accepted therapeutic management comprises comprehensive intensive care, early administration of penicillin in combination with clindamycin, and surgical intervention. The use of intravenous immunoglobulins (IVIG), elimination methods and hyperbaric oxygen are under discussion. The slight increase in cases and ineffective prevention require rapid assessment of diagnosis and adequate treatment as a protracted course of the condition is connected with a high mortality rate.

Cognitive performance in people with Parkinson's disease and mild or moderate depression: effects of dopamine agonists in an add-on to L-dopa therapy.

In a randomized prospective multi-centre study, we evaluated the cognitive performances of a group of 41 non-demented patients, all with advanced Parkinson's disease (PD) and a current depressive episode, in whom the effects of pramipexole (PPX) and pergolide (PRG) in an add-on to l-dopa therapy were also studied and published with regard to motor symptoms of PD, motor complications and depression. The Trail Making Test, the Stroop test and four subtests (arithmetic, picture completion, digit symbols and similarities) of the Wechsler Adult Intelligence Scale-Revised were performed prior to and 8 months after the administration of either PPX or PRG. We found no statistically significant difference between the two tested drugs or between the first and the last visit in any of the above-listed neuropsychological tests. All patients' motor outcomes significantly improved and we conclusively demonstrated the anti-depressive effect of PPX. The dissociation of dopaminomimetic effects on the different tested domains indicates that there are different pathological mechanisms of cognitive, motor and affective disturbances in advanced PD patients. In our non-demented group of fluctuating depressed PD subjects, both PPX and PRG administration in combination with l-dopa were safe in terms of the effect on cognitive performance.

Long term and repeated electron microscopy and PCR detection of Borrelia burgdorferi sensu lato after an antibiotic treatment.

The diagnosis of Lyme disease in 18 patients has been proved by detection of Borrelia burgdorferi sensu lato when using immunoelectron microscopy or detecting its nucleic acid by PCR in the plasma or the cerebrospinal fluid. The positive results occurred in the plasma or in the cerebrospinal fluid in the period of 4-68 months after an antibiotic treatment. The typical clinical manifestations of Lyme disease were observed in 9 patients and non-specific symptoms in another 9 patients. According to presented results we can recommend repeated examination using PCR of the plasma and other biological specimens in the individuals with persistent or recurring complaints after an acute form of Lyme disease and its antibiotic treatment. Also examination of the cerebrospinal fluid with non-specific symptoms and simultaneously displayed pathology electroencephalogram and/or magnetic resonance imaging findings can be advantageous.

Pramipexole and pergolide in the treatment of depression in Parkinson's disease: a national multicentre prospective randomized study.

An 8-month multicentre prospective randomized study aimed at comparing the effects of dopamine receptor agonists pramipexole (PPX; Mirapexin) and pergolide (PRG; Permax) as add-on to L-dopa therapy on depression [Montgomery and Asberg Depression Rating Scale (MADRS)] in 41 non-demented patients (25 men, 16 women) suffering from both mild or moderate depression and advanced Parkinson's disease (PD). The assessment was performed by a blinded independent observer. Motor symptoms (UPDRS III), motor complications (UPDRS IV), activities of daily living (UPDRS II and VI) and depressive symptoms as measured by Self - Rating Depression Scale by Zung were evaluated in an open-label design. The average value of Zung scores decreased significantly in both groups with no statistical difference between both groups. A significant decrease in the average value of MADRS scores was present only in the PPX group. The average UPDRS scores decreased significantly with no statistical difference between both groups at the comparable average total daily dose of both preparations. In both cases, the total daily dose of L-dopa decreased significantly but the decrease was statistically more pronounced in the PRG group. Our results demonstrate the antidepressant effect of PPX in patients with PD while we can't make any conclusions with regard to antidepressant effect of PRG.

Administration of antibiotics before admission in patients with meningococcal disease.

PURPOSE: To determine whether parenteral or oral antibiotics given before admission to a regional hospital with a special intensive care unit (ICU) reduce the case fatality rate in patients with meningococcal disease. DESIGN: Prospective analysis of 164 consecutive patients with meningococcal disease admitted to 5 regional hospitals in the Czech Republic between August 1996 and October 2001. Main outcome measure was number of deaths from meningococcal disease. Fisher's exact test was used for statistical analysis. MAIN FINDINGS: Nine out of 116 patients (8%) given antibiotics before admission died, compared with five deaths in 48 patients (10%) admitted without such a treatment (p = 0.55). None of 19 patients given oral or combined oral and parenteral pre-admission antibiotics died. CONCLUSION: Parenteral and probably also oral antibiotics given before admission to a regional hospital and an adequate treatment of shock can reduce the case fatality rate from meningococcal disease.

[Indications for percutaneous endoscopic gastrostomy in patients with disorders of the nervous system]. / Indikace perkutánní endoskopické gastrostomie u nemocných s poruchou nervového systému.

Percutaneous endoscopic gastrostomy (PEG) is an efficient endoscopic method that ensures enteral nutrition for a longer period of time in patients who cannot take food per os. This method is also indicated in patients suffering from disorders of the central or peripheral nervous system which developed suddenly, such as a stroke or craniocerebral injuries, or gradually, such as amyotrophic lateral sclerosis (ALS), dementia, and multiple sclerosis. It has become common practice in the cooperation between neurologists and a gastroenterologists to use PEG in patients hospitalized in a neurological ward with encephalomalacy and haemorrhage, or craniocerebral injuries (after the patient recovers from the acute stage of the disease and is transferred to a neurological ICU), as well as in patients with ALS in a progressive stage. We gradually extend the indications of PEG for other patients with neurological disorders such as patients suffering from dementia, progressive multiple sclerosis, Parkinson's disease, and progressive polyneuropathy. Of 62 patients hospitalized in a neurological ward during a period of 4.5 years, 56 patients suffered from sudden disorders of the nervous system (strokes and craniocerebral injuries) and 6 patients had gradually progressing neurological diseases (ALS, multiple sclerosis, Parkinson's disease, dementia, and polyneuropathy).

[Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis]. / Perzistence Borrelia burgdorferi sensu lato u pacientu s lymeskou borreliózou.

In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia burgdorferi sensu lato by detection of the causal agent by immune electron microscopy or of its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months. Clinical manifestations common in Lyme borreliosis were present in only half the patients, in the remainder non-specific symptoms were found. In nine subjects with confirmed Borrelia burgdorferi sensu lato in the cerebrospinal fluid the cytological and biochemical finding was normal. Examination of antibodies by the ELISA method was negative in 7 of 18 patients during the first examination and in 12 of 18 during the second examination. In all negative examinations the specific antibodies were assessed by the Western blot or ELISA method after liberation from the immunocomplexes. In the authors' opinion it is advisable to examine repeatedly plasma and other biological material from potentially affected organs by PCR and subjects with persisting or relapsing complaints after the acute form of Lyme borreliosis as well as to examine cerebrospinal fluid in case on non-specific symptoms and concurrent pathic EEG or MR findings.

[Criteria for evaluation of immunoblots using Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto for diagnosis of Lyme borreliosis]. / Kritéria hodnocení imunoblotu v diagnostice lysmké borreliózy pri pouzití Borrelia afzelii, Borrelia garinii a Borrelia burgdorferi sensu stricto.

The immunoblot was prepared from genotypes Borrelia afzelii (KC 90), Borrelia garinii (M 192) and Borrelia burgdorferi sensu stricto (B 31). Sera of 63 patients with different forms of Lyme borreliosis were examined and 40 healthy donors in the endemic area of the disease. In class IgM in the group of patients significantly more frequently antibodies against OspC, p39, p41 B. afzelii, p39, p41, p66, p83 B. garinii and OspC1, OspA, B. burgdorferi sensu stricto were found. In class IgG there were antibodies against p39, p41, p93 B. afzelii, p14, p41, p93 B. garinii and OspA, OspC p93 B. burgdorferi sensu lato. Based on the assembled results by means of discrimination analysis and logistic regression the most suitable combinations of antigens for evaluation of immunoblots in different genotypes were determined. Furthermore evaluation was suggested using a combination of antigens of several genotypes which led to an increased sensitivity and specificity of the immunoblot. Tables were prepared for easier evaluation of newly examined sera samples.

[Importance of the immunoblot test in the diagnosis of Lyme borreliosis]. / Význam imunoblotu v diagnostice lymské boreliózy.

Using a commercial kit for the examination of recombinant immunoblot the authors examined sera of 85 patients with direct evidence of Borrelia burgdorferi sensu lato in serum or cerebrospinal fluid or patients with typical dermal form of borreliosis. The results were compared with results of assessment of specific antibodies by the ELISA test. The authors investigated the importance of antibody formation against different antigens in different clinical forms of the disease. The specificity of the investigated kit was higher than that of the ELISA test, the sensitivity was lower. Examination of the immunoblot was negative in 8.8% patients with direct evidence of the causal agent of Lyme borreliosis. A suitable configuration of the recombinant immunoblot for use in Europe where three genotypes of Borrelia burgdorferi sensu lato are found requires further investigation. Another problem are criteria for evaluation of positivity of the blot in Europe.

Comparative studies of two potency tests for antirabies serum: neutralization test in mice (MNT) and rapid fluorescent focus inhibition test (RFFIT).

At our institute the mouse neutralization test (MNT) was replaced by the rapid fluorescent focus inhibition test (RFFIT). Therefore, RFFIT results were statistically analyzed for their percentage limits at the 95% confidence level and were compared with those of the MNT. An anti-rabies immunoglobulin of human origin and the WHO standard serum were assayed on 20 consecutive days to determine the reproducibility of the RFFIT. Sera of 60 vaccinees were assayed for rabies antibodies by the RFFIT and MNT. The titers as well as the international units per ml showed a correlation of 0.95 and 0.87 respectively. In accordance with previous publications by other authors it was found that the RFFIT is a significantly better reproducticible test system than the MNT and excellently correlates with the latter method.

Enhancement of Lewis lung carcinoma by the concomitant infection of the host with herpes simplex virus type 1 and type 2.

Infection with herpes simplex virus (HSV) enhances the growth of Lewis lung tumor (LLT). Infection with HSV-1 enhances as efficiently as HSV-2. A significant acceleration of primary tumor formation was obtained. In mice, lung metastases were increased in number and size above the controls. Enhancement occurred only with high doses of infectious virus and was also obtained only when the virus infection was at a site close to that of the tumor implantation. This finding, in addition to the lack of antigenicity of LLT, is interpreted as arguing against an immunologic mechanism of the phenomenon.

Augmented immunogenicity of Lewis lung carcinoma by infection with herpes simplex virus type 2.

In vitro, LLT cells sustain HSV-2 replication without evidence of lysis. Simultaneously, multiplication of the cells is stimulated. These xenogenized cells were tested for their immunopotentiating capacity: three-step immunization with xenogenized viable cells conferred significantly augmented transplantation resistance to a challenge graft with 4 x 10(4) intact LLT cells. Latency periods preceding tumor formation were increased and 15% of the mice failed to form primary tumors. Metastasis was likewise decreased and 25% of the mice had healthy lungs. Immunopotentiation, however, did not suffice to significantly protect against a challenge with 6 x 10(4) intact cells. The presence of virus-specific neoantigens on HSV-2-infected viable cells was demonstrated by the progressively increasing number of rejections of 4 x 10(4) xenogenized cells during the successive immunization steps. Immunization with non-viable LLT cells did not augment resistance to challenge.

Herpes virus infection as a cofactor in carcinogenesis: supernatants of herpes virus type 1- and type 2-infected cell cultures contain a cell growth-stimulating factor.

Some cell cultures synthesize a mitogenic factor (DNASF) following their lytic infection with either HSV-1 or HSV-2 which is shed into the medium. Of six cell lines permissive to HSV infection tested, three produce DNASF (CV1, HF, MCA) and a significantly higher (3H)-TdR incorporation was obtained with indicator cells growing in supernatants of virus-infected cells (SupV+) than in the corresponding supernatants of sham-treated cells (SupV-). Sometimes the values obtained with SupV+ exceed those obtained with cell controls, which demonstrates that DNASF is not simply a nutritional factor. Other cell lines do not produce DNASF (HeLa, Wistar and probably REF) and SupV+ frequently inhibit cell growth. DNASF is genetically nonspecific and all indicator cells tested were stimulated by supernatants which contain the mitogen, including the nonpermissive DC-3F cells. The quantity of DNASF induced is directly proportional to the number of cells in the culture and its production peaks when the lysis of the cells is complete. It is not accumulated inside the cells.

[Herpes simplex virus (hsv-1 and hsv-2) infection: its clinical and oncogenic properties (author's transl)]. / Die Herpes-simplex-Virus-(HSV-1-und HSV-2-) Infektion- ihre klinische und onkogene Bedeutung.

The epidemiology and clinical features of diseases caused by the herpes simplex virus, are reviewed and recent results are discussed which give an insight into the complex mechanism of primary and chronic, recurrent HSV-1 infections. Immunological reactions in HSV infection and data concerning the oncogenic potential of HSV-1 and -2 are dealt with. Furthermore, current therapeutic possibilities are outlined.

An investigation into the relationship of the immunological status of Herpes simplex virus type-2 (HSV-2) infected animals and the tumor-enhancing property of this infection.

In order to compare the state of immunity of HSV-2 infected animals with their sham treated counterparts, several in vivo methods for cell-mediated immunity in addition to the stimulation in vitro of mouse splenocytes with PHA were performed. The grafting of C 3 H mice with heterologous skin and the paw test with PHA showed no noticeable difference between the two groups. The tuberculin test performed on guinea pigs infected via the footpads, permitted the detection of a slightly impaired reactivity of the virus-infected animals. The PHA assay in vitro showed reduced stimulation indices with cultures of splenocytes from virus-infected mice about 10 days p.i. These reduced stimulation indices are the result of 1. a low 3 HTdR incorporation by the PHA stimulated cultures and 2. of a relatively high 3 HTdR uptake by the unstimulated control cultures. An interpretation of these results is suggested. Finally, the possible implications of a shortlived impaired cell-mediated immunity on the tumour enhancing property of the HSV-2 infection, which has been described in previous publications, are discussed.

The phosphorylation of histones in herpes virus infected Vervet-monkey kidney cells (CV-1).

The phosphorylation of histones of the F1 group and of fraction F2a2 is stimulated in monkey kidney cells (CV-1) to almost two times the control values 14 hours after their infection with herpes virus, type 2. At the same time high amounts of viral DNA are produced. It seems very likely that the stimulated phosphorylation of these histone fractions is a prerequisite for the enhanced synthesis of DNA and possibly also RNA.