Mikko Järvinen

Human spleen cysteineproteinase inhibitor: Purification, fractionation into isoelectric variants and some properties of the variants

The papain inhibitor from human spleen was purified by extraction in isotonic sucrose, acetone fractionation, papain-Sepharose affinity chromatography and gel filtration on Sephadex G-50. The purified inhibitor was fractionated by electrofocusing into four major isoelectric variants with pI values of 4.7, 5.0, 6.0 and 6.5. These variants can be classified into two groups: the acidic type, comprising the variants with pI 4.7 and 5.0, and the neutral type, comprising the variants with pI 6.0 and 6.5. The following properties distinguish the two types: 1. Immunological properties: antibodies raised against either of the neutral variants precipitated both of these, but not the acidic variants. The antiserum against the human epidermal cysteineproteinase inhibitor precipitated the acidic variants, but not the neutral variants. 2. Molecular size: two-dimensional electrophoresis of the purified inhibitor gave molecular weights of 11400 for the acidic variants and 12000 for the neutral variants. The pI 6.0 variant contained two compounds with molecular weights of 12000 and 12800. 3. Enzyme spectrum: human cathepsin B was inhibited by the acidic type, while the neutral type was a poor inhibitor. Both types inhibited cathepsin H, papain, ficin and bromelain, although the inhibition of bromelain did not exceed 70%. Human cathepsin D, bovine trypsin and chymotrypsin and porcine elastase were not inhibited by either type.

Reduced cystatin B activity correlates with enhanced cathepsin activity in progressive myoclonus epilepsy.

BACKGROUND: Loss-of-function mutations in the gene encoding cystatin B (CSTB) underlie an inherited neurodegenerative disorder, progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). CSTB is an inhibitor of several papain-family cysteine proteases, the lysosomal cathepsins. Its physiological function and the molecular pathways leading to the clinical EPM1 phenotype are unknown. AIM: To elucidate the role of CSTB and different cathepsins in pathogenesis of EPM1. METHOD: We determined the total papain inhibitory (cystatin) and papain-like (cathepsin) activity as well as specific activities of cathepsins B, H, L and S in lymphoblastoid cells of EPM1 patients, carriers and controls. RESULTS: In EPM1 patients, who express reduced levels of CSTB mRNA, the papain inhibitory activity was significantly decreased or absent. This reduction was correlated with significant increase in general cathepsin activity. The increase in cathepsin B, L and S activities was highly significant, whereas the increase in cathepsin H activity was not. CONCLUSIONS: This is the first demonstration of cysteine protease activity being regulated by CSTB activity in a biological context. The effects of decreased CSTB activity in EPM1 pathogenesis may, at least in part, be mediated by cathepsins through increased activity of cathepsins S and L.

Human cystatins in normal and diseased tissues--a review.

Immunohistochemica and quantitative immunochemical methods were used to demonstrate the presence of two cysteine proteinase inhibitors, cystatins A and B, in normal and diseased tissues. Cystatin A is expressed in squamous epithelia, neutrophil granulocytes, and dendritic reticulum cells of the lymphatic tissues. Its concentration is increased in inflammatory skin diseases and decreases after the malignization of squamous epithelia. Cystatin B is seen in wet squamous epithelia, and in the cells of monocyte-macrophage series, where its concentration varies depending on the activation state of the cells. In the malignant keratinocytes cystatin B follows the behaviour of cystatin A.

Purification and some characteristics of two human serum proteins inhibiting papain and other thiol proteinases

Human serum contains 7 well characterized proteinase inhibitors with more or less broad specificity to serine proteinases [ 1,2]. In addition, two inhibitors not inhibiting serine proteinases have been described: fl,-collagenase inhibitor [3] and cYz-thiol proteinase inhibitor [4]. The partially purified cuz-thiol proteinase inhibitor has been shown not to be identical with the known inhibitors of serine proteinases. Its properties are also quite different from those of the human epidermal thiol proteinase inhibitor, purified in our laboratory [5,6]. Here I describe the purification of two thiol proteinase inhibitors with (ILLand CQ-mobilities from human serum. The purified inhibitors resemble each other in their immunological properties and inhibiting spectra, but have different charges and molecular sizes. The inhibitor with cYz-mobility seems to be identical with the thiol proteinase inhibitor in [4]. A preliminary note of this work has been published [7].

Demonstration of immunoreactive acid cysteine-proteinase inhibitor in reticulum cells of lymph node germinal centres.

SummarySeven human lymph nodes showing different types of reactive change, were examined for the presence of acid cysteine-proteinase inhibitor (ACPI) by the peroxidase-antiperoxidase method. A clear positive reaction was found in the germinal centres. The staining pattern indicated immunoreactivity of the dendritic reticulum cells, but the possibility that other cells, particularly histiocytic reticulum cells, may also react with antiserum raised against ACPI cannot be excluded.

Atrial natriuretic polypeptides in the specific atrial granules of the rat heart: immunohistochemical and immunoelectron microscopical localization and radioimmunological quantification

Atria of several mammalian species contain atrial natriuretic polypeptides (ANP) with natriuretic, diuretic, and vasodilating activity. In the present studies ANP were localized and quantitated in different parts of the heart by immunocytochemical and radioimmunological methods. The concentration of immunoreactive ANP as determined by quantitative radioimmunoassay in rat heart atria was a follows (ng/mg, mean +/- SD, n = 5): right auricle (688 +/- 156), left auricle (556 +/- 156), right atrium (334 +/- 60), and left atrium (93 +/- 36). The staining intensities in immunohistochemical localizations were consistent with the quantitative data. The location of the peptides was sarcoplasmic and granular. The highest concentration of ANP was found in the perinuclear area of the atrial myocyte sarcoplasm, but some staining was also seen in the periphery of the cells. The indirect immunoelectron microscopical gold method showed that ANP are located in the specific atrial granules supporting previous findings.

Cysteine proteinase inhibitors in psoriatic epidermis

SummaryHuman psoriatic epidermis and scales were demostrated to contain two antigenically separate cysteine proteinase inhibitors, one acidic with an isoelectric point of 4.7–5.0 (ACPI) and one neutral with an isoelectric point of 6.0–6.5 (NCPI), while normal epidermis contains only ACPI. The total papain (cysteine proteinase) inhibiting activity of the psoriatic epidermis as calculated per mg protein was higher than that in normal epidermis. Both ACPI and NCPI were localized immunocytochemically, mainly in the highest spinous cell layers with less activity in the parakeratotic cells and lower layers of spinous cells. Basal cells were essentially negative.

Localization of the human SH-protease inhibitor in the epidermis: Immunofluorescent studies

Human epidermis contains a low molecular weight SH-protease inhibitor (Human Epidermal Inhibitor = HEI), whose epidermal localization was performed with the indirect immunofluorescence method. The fluorescence was most intensive in the cytoplasms of epidermal cells, often occurring perinuclearly. The fluorescent material in the frozen sections was often finely granular and occasionally extended outside the cytoplasm, while the fluorescence in fixed sections was more uniform, but weaker. Stratum basale generally stained poorly or not at all, as did also stratum lucidum. Stratum corneum stained fairly intensively throughout. In addition to fixation, the outcome of staining was also affected by the thickness of the epidermis, particularly stratum corneum. The significance of this inhibitor for the differentiation of epidermal cells and the keratinization of epidermis has therefore been discussed, and the authors assume it to be of considerable significance in these processes.

Cystatin a-like immunoreactivity is widely distributed in human brain and accumulates in neuritic plaques of alzheimer disease subjects

The cellular localization of cystatin A, an endogenously occurring inhibitor of lysosomal thiol proteases (cathepsins B, H, L and S), was studied immunohistochemically in human postmortem brain using the peroxidase-antiperoxidase method. Both polyclonal and monoclonal antibodies to cystatin A were employed. Western blot analysis revealed one molecular form of the inhibitor in human brain extracts. Its molecular weight was about 13,000. Immunostaining appeared in a sizeable population of neurons and a few cells surrounding cerebral blood vessels (pericytes). In Alzheimer disease subjects cystatin A was found in many neuritic plaques. Possible functional consequences with regard to a role of cystatin A in the inhibition of the Alzheimer amyloid precursor protein (APP)-clipping enzyme, cathepsin B, are discussed.

Immunohistochemical localization of l-Ornithine decarboxylase in developing rat brain

l‐Ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis and a marker enzyme of tissue proliferation and maturation, was localized immunocytochemically in the developing rat central nervous system. It can be noted that the distribution of the enzyme protein underlies temporal alterations. Conclusions are drawn from the location of the enzyme and possible functional roles played by ornithine decarboxylase in discrete brain areas.