Alopecia areata in a rhesus monkey (Macaca mulatta)

BACKGROUND: A 14-year-old female rhesus monkey (Macaca mulatta) of Chinese origin has been suffering from alopecia universalis since childhood. METHODS: Recently, the health status of the animal was recorded comprehensively by detailed clinical examination including hematology and serology supplemented by histological and immunohistochemical investigations of skin biopsies and molecular biological techniques to clarify the causes of the persistent hair loss. RESULTS AND CONCLUSIONS: The hairless gene (hr) nonsense mutation was ruled out by polymerase chain reaction and by sequencing of the corresponding gene. Histological examinations revealed a prominent chronic lymphocytic perifolliculitis and folliculitis affecting anagen stage hair follicles as well as miniaturized hair follicles. Immunohistochemistry using the antibodies CD3, CD20 and CD4 confirmed the diagnosis of a T-cell-mediated autoimmune disease resembling alopecia areata universalis in humans.     

Invasive aspergillosis in a putty-nosed monkey (Cercopithecus nictitans) with adrenocortical Cushing's syndrome

Background  An 18‐year‐old captive female putty‐nosed‐monkey (Cercopithecus nictitans) with a history of long‐term infertility and hyperglucocorticism was euthanized because of perforating thoracic trauma induced by group members and subsequent development of neurological signs. Methods  Complete necropsy and histopathological examination of formalin‐fixed tissue samples was carried out. Results  The monkey showed invasive pulmonary and cerebral infection with Aspergillus fumigatus together with adrenocortical neoplasia and signs of Cushing’s syndrome, such as alopecia with atrophic skin changes, evidence for diabetes mellitus and marked immunosuppression. Conclusions  Spontaneous endocrinopathies are rarely described in non‐human primates. Here we report the first case of spontaneous adrenocortical hyperglucocorticism predisposing to systemic aspergillosis in a putty‐nosed monkey.     

Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.     

Ancient metalloenzymes as possible markers in molecular archaeology

The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.     

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.     

Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example

Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of approximately 500 microm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of < or =42 microm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL(-1) was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/cell formation.     

Construction and analysis of a novel peptide tag containing an unnatural N-glycosylation site

The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal "tail" sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra N-glycan moieties by simply adding a peptide tag sequence as small as 22 amino acids.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.