The Influence of Modern Neurophysiology on the Previous Definitions of "Segment" and "Interference Field" in Neural Therapy.

BACKGROUND: In neural therapy, local anesthetics are injected for diagnostic and therapeutic purposes. In this process, therapy makes use of the regulatory functions and plastic properties of the nervous system, especially its autonomic part. Up until now, a distinction has been made between "local/segmental neural therapy" and "interference field therapy." This division dating back to the middle of the last century was based on the assumption that anatomical and clinical segments were identical. However, this is only true for the projection symptoms, which are limited to metamerism. All pathophysiological processes beyond this segment were called "interference field events" ("outside of any segmental order" and "not explainable by neuroanatomical circuitry"). SUMMARY: However, modern neurophysiology no longer recognizes segmental boundaries, taking into account the occurrence of cross-segmental sensitization processes, neuroplastic changes, immune processes, and neurogenic inflammation. In addition, new insights into neuroanatomical circuitry have also contributed to segmental expansion. Thus, in recent years, much of the interference field activity has been assigned to an "extended" segment; however, even there, no segment boundaries can be defined. Thus, the former definition of the interference field effect (considered to be outside any segmental order) is considered obsolete. Nowadays, interference fields are called "neuromodulatory triggers." They can act anywhere, both locally and fairly distant, and even systemically. KEY MESSAGES: Thus, it is no longer tenable to classify interference field therapy as "unscientific" and "not recognized" while local and segmental neural therapy is being scientifically recognized. In the work at hand, the interference fields discovered by the Huneke brothers become scientifically definable as "neuromodulatory triggers" by showing that clinically and pathologically, hardly any segmental boundaries exist.

OPN deficiency results in severe glomerulosclerosis in uninephrectomized mice.

Osteopontin (OPN) expression has been reported to be elevated in experimental models of renal injury such as arterial hypertension or diabetic nephropathy finally leading to focal segmental glomerulosclerosis (FSGS). FSGS is characterized by glomerular matrix deposition and loss or damage of podocytes that represent the main constituents of the glomerular filtration barrier. To evaluate the role of OPN in the kidney we investigated WT and OPN knockout mice (OPN-/-) without treatment, after uninephrectomy (UNX), as well as after UNX and desoxycorticosterone acetate (DOCA)-salt treatment with respect to urine parameters, glomerular morphology, and expression of podocyte markers. OPN-/- mice showed normal urine parameters while a thickening of the glomerular basement membrane was evident. Intriguingly, following UNX, OPN-/- mice exhibited prominent FSGS, proteinuria, and glomerular matrix deposition. Electron microscopy revealed bulgings of the glomerular basement membrane and occasionally an effacement of podocytes. After UNX and DOCA-salt treatment, severe glomerular lesions as well as proteinuria and albuminuria were seen in WT and OPN-/- mice. Moreover, we found a reduction of specific markers such as Wilm's tumor-1, podocin, and synaptopodin in both experimental groups indicating a loss of podocytes. Podocyte damage was accompanied by increased number of Ki-67-positive cells in the parietal epithelium of Bowman's capsule. We conclude that OPN plays a crucial role in adaptation of podocytes following renal ablation and is renoprotective when glomerular mechanical load is increased.

Effect of different artificial tears against desiccation in cultured human epithelial cells.

BACKGROUND: A large number of artificial tears is widely used to treat dry eye symptoms. To test the efficacy of these drugs independent of individual parameters in vitro models are required. As described previously, we employed a reproducible in vitro cell culture system to evaluate the desiccation protection capability of some artificial tears. In THE PRESENT PAPER DATA IS PRESENTED OF ANOTHER SET OF PHARMACEUTICAL AGENTS. MATERIAL/METHODS: Conjunctival epithelial cell line Chang 1-5c-4 (series 1) and the corneal cell line 2.040 pRSV-T (series 2) were cultured under standard conditions. Confluent cells were wetted for 20 min with artificial tears (Arufil Uno, Arufil, Lacrimal, Lacophthal sine, Siccaprotect, Tears Again, Vidisept EDO, Vistil, Wet Comod) or PBS as a control. After exposure to a constant air flow for 0, 15, 30 and 45 minutes respectively, cells were incubated with the vital dye alamarBlue. Subsequently, absorption of the oxidised form of the dye was assessed using an ELISA-Reader. RESULTS: Cell best survival rates in series 1 after 15 min were found for Lacrimal (0.89), Wet Comod (0.84) compared to PBS (0.66) and in series 2 for Vidisept EDO (0.57) and Lacrimal (0.56) compared to PBS (0.01). After 45 min highest survival was seen in series 1 for Lacrimal (0.46) and Lacophthal sine (0.36) compared to PBS (0.33) and in series 2 for Lacrimal (-0.06) and Arufil (-0.16) compared to PBS (-0.23). CONCLUSIONS: Both cell lines tested showed different susceptibility towards desiccation and the artificial tears showed differences in preventing cells from desiccation.

Morphology and migration of podocytes are affected by CD151 levels.

CD151, a member of the tetraspanin family of membrane proteins, is crucially involved in the formation of the glomerular filtration barrier in humans and mice. However, the role of CD151 in podocytes has not been investigated so far. In the present study, we utilized a conditionally immortalized mouse podocyte cell line to characterize CD151 in podocytes and to examine the consequences of manipulating CD151 expression levels. Mouse podocytes endogenously express CD151 as determined by RT-PCR and Western blotting. GFP-CD151 fusion protein localized to the cell membrane, to cell protrusions and cell-cell contacts, colocalizing with actin, ß(1)-integrin, zonula occludens-1, and CD9. The expression of GFP-CD151 in cultured podocytes resulted in a marked increase in the presence of thin arborized protrusions (TAPs). TAPs are distinct from filopodia by increased length, protein composition, branched morphology, and slower dynamics. Furthermore, the migration rate of pEGFP-CD151-transfected podocytes was reduced in a wound assay. Fluorescence recovery after photo bleaching measurements revealed a half-time of 3 s for GFP-CD151 consistent with a high mobility of CD151 in the membrane and cytosol. CD151 knockdown in podocytes reduced ß(1)-integrin expression and podocyte cell area, indicating diminished adherence and/or spreading. Our results indicate that CD151 importantly modulates podocyte function.

Laser scanning confocal microscopy for conjunctival epithelium imaging.

BACKGROUND: Conjunctival disorders may adversely affect tear film and promote/induce the development of sicca syndrome (also known as Sjögren's syndrome). The basic diagnostics of sicca syndrome are slit lamp examination and functional tests (such as the Schirmer test, break-up time, or fluorescein/rose bengal staining). However, morphological analysis requires time and effort, both in terms of technical equipment and labor, and the results are not available immediately. In contrast, when using laser scanning confocal microscopy (LSCM), the anatomy and morphology of the conjunctival epithelium may be evaluated in vivo during the clinical examination. MATERIAL AND METHODS: We examined the conjunctival epithelium of 23 subjects with healthy eyes using LSCM. We compared intraindividual morphological patterns of normal conjunctival epithelium derived from the Heidelberg Retina Tomograph II - Rostock Cornea Module (HRTII-RCM) with those from impression cytology. All examinations were performed on the conjunctiva bulbi at the 12 o'clock position, 2 mm from the limbus corneae. RESULTS: LSCM and impression cytology examine the conjunctival epithelium from identical perspectives. This facilitates an intraindividual comparison of morphological patterns. In addition, artifact detection and the mapping of light/dark pattern recognition of the LSCM to the microscopy of the impression cytology were reliable. LSCM allows in vivo discrimination of non-secretory from secretory cells in conjunctival epithelium. Non-secretory epithelium shows dark, light and bright cytoplasm of epithelial cells on LSCM, in contrast to impression cytology. Nucleoplasmic ratio ranged from 1:1 to 1:4. Shape, size and interior structure were reliable criteria to distinguish goblet cells from non-secretory cells. The interior structure of the goblet cells showed dark or highly reflective bright homogeneous textures. CONCLUSION: LSCM is a feasible method for examining the morphology of conjunctival epithelium using non-invasive in vivo imaging. Morphological criteria for squamous metaplasia of the conjunctiva in sicca syndrome are already known from cytology, and can be used in almost the same manner in LSCM. The separation of epithelial microcysts from small goblet cells is difficult with LSCM. Finally, the clinical application of LSCM in the staging of sicca syndrome has to be evaluated in future studies.

Immunohistochemical expression of caspase-1 and -9, uncleaved caspase-3 and -6, cleaved caspase-3 and -6 as well as Bcl-2 in benign epithelium and cancer of the prostate.

Activation of caspases is an essential prerequisite for induction of apoptosis. In many tumors caspases are down-regulated, while anti-apoptotic Bcl-2 is up-regulated. To elucidate their putative role in prostate cancer (PCa) we determined the expression of different caspases and Bcl-2 in benign prostate epithelium (BPE) and PCa. Paraffin-embedded prostate whole mounts were cut (4 µm) and investigated immunohistochemically using monoclonal antibodies against caspase-1 and -9, uncleaved caspase-3 and -6, cleaved caspase-3 and -6, and Bcl-2. In BPE all caspases were localized to the cytoplasm of glandular cells. In PCa we found a statistically significant reduction in cleaved caspase-3 and -6 compared to the levels in BPE. The Bcl-2 protein was detected in the basal compartment of epithelial gland cells, but no immunostaining was noted in PCa. The decreased immunoreactivity of activated caspases probably indicates an alteration in post-translational cleavage that may play an important role during PCa progression.

Alterations of the podocyte proteome in response to high glucose concentrations.

Diabetic nephropathy is one of the most common complications of diabetes mellitus and the leading cause of end-stage renal disease. A reduction in podocyte number has been documented in the kidneys of these patients. To identify the molecular changes in podocytes that are primarily caused by high glucose (HG) concentrations and not by secondary alterations (e.g. glomerular hypertension), we investigated the protein expression profiles in a podocyte cell line under long-term HG exposure (30 versus 10 mM for 2 wk). Proteins were separated by 2-DE, and we identified 39 different proteins in 48 spots that were differentially regulated by more than twofold in response to HG concentrations using MALDI-TOF MS and MASCOT software. These proteins belong to several protein classes, including cytoskeletal proteins and specific annexins (annexins III and VI). Downregulation of annexins III and VI by HG concentrations was confirmed by qRT-PCR, Western blot, and immunostaining, and was also observed in glomeruli of kidney biopsies from patients with diabetic nephropathy. Our data demonstrate that HG concentrations per se are sufficient to strongly modify the protein expression profile of podocytes, the analysis of which contributes to the identification of novel targets involved in diabetic nephropathy.

Altered expression of tumor protein D52 regulates apoptosis and migration of prostate cancer cells.

Tumor protein D52 (TPD52) is a protein found to be overexpressed in prostate and breast cancer due to gene amplification. However, its physiological function remains under investigation. In the present study, we investigated the response of the LNCaP human prostate carcinoma cell line to deregulation of TPD52 expression. Proteomic analysis of prostate biopsies showed TPD52 overexpression at the protein level, whereas its transcriptional upregulation was demonstrated by real-time PCR. Transfection of LNCaP cells with a specific small hairpin RNA giving efficient knockdown of TPD52 resulted in significant cell death of the carcinoma LNCaP cells. As demonstrated by activation of caspases (caspase-3 and -9), and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with enhanced green fluorescence protein-TPD52 or a specific small hairpin RNA. Enhanced green fluorescence protein-TPD52 overexpressing cells showed an increased proliferation rate, whereas TPD52-depleted cells showed the reverse effect. Additionally, we demonstrate that exogenous expression of TPD52 promotes cell migration via alphav beta3 integrin in prostate cancer cells through activation of the protein kinase B/Akt signaling pathway. From these results, we conclude that TPD52 plays an important role in various molecular events, particularly in the morphological diversification and dissemination of prostate carcinoma cells, and may be a promising target with respect to developing new therapeutic strategies to treat prostate cancer.

Dominance of cytokine- over FasL-induced impairment of the mitochondrial transmembrane potential (Deltapsim) in the pancreatic beta-cell line NIT-1.

Mitochondria of pancreatic beta-cells are potential targets of intrinsic and extrinsic apoptotic pathways in the autoimmune pathogenesis of type 1 diabetes. We aimed to investigate whether cytokine- and FasLigand (FasL)-induced apoptosis is associated with impaired mitochondrial transmembrane potential (Deltapsim) in the pancreatic beta-cell line NIT-1. NIT-1 cells were exposed to the interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma) cytokine combination to induce apoptosis in vitro. Low concentrations of cytokines resulted in Deltapsim impairment, and increasing concentrations had only a minor additional effect. Treatment with the inducible nitric oxide synthase (iNOS) inhibitor Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME) prevented cytokine-mediated Deltapsim impairment, implying that cytokines affect Deltapsim via nitric oxide. The broad-spectrum caspase inhibitor Z-VAD(Ome)-FMK (ZVAD) revealed dichotomic actions. In the presence of ZVAD, cytokine-induced nitrite generation was increased but cell death and Deltapsim impairment were reduced. Deltapsim impairment was also reduced by inhibitors of caspases 1, 6 and 8. Induction of Fas by IL-1beta/IFN-gamma coupled with activation by Super-FasL augmented cytokine-induced cell death. We observed a clear dominance of cytokine- over FasL-induced effects on Deltapsim. Our findings show that IL-1beta/IFN-gamma cytokines have a strong effect to impair Deltaym and prime beta-cells for apoptosis via the intrinsic pathway mediated by iNOS and caspases. Furthermore, at least in NIT-1 cells, the extrinsic FasL/Fas pathway has only a minor additive effect on cytokine-induced Deltapsim impairment.

Evaluation of apoptotic cells and immunohistochemical detection of FAS, FAS-L, Bcl-2, Bax, p53 and c-Myc in the skin of patients with chronic venous leg ulcers.

In the present study we were interested, if apoptosis plays a role in the surrounding skin of venous ulcers, where microcirculatory disorders were already observed. For this purpose laser Doppler flow and partial oxygen pressure were measured in 17 patients at the ulcer edge, the transitional area of the lower leg and the thigh. Subsequently biopsies were taken from the respective sites and subjected to terminal deoxynucleotidyl transferase labelling (TUNEL) and immunohistochemistry using antibodies to determine the protein expression of Fas, Fas-L, Bax, Bcl-2, p53 and c-Myc. Laser Doppler flow was increased and transcutaneous oxygen partial pressure was decreased, with significant differences at the ulcer edge and the lower leg compared to the thigh. The skin biopsies did not show any differences when labelling for apoptotic cells. Keratinocytes of basal and spinous layer stained with antibodies against Fas, Fas-L and Bax in all probes of the three sites. c-Myc and p53 were negative in all keratinocytes of the skin probes. However, staining with Bcl-2 was significantly decreased at the ulcer edge in comparison to the lower leg and the thigh (p=0.017). Our study revealed that a disturbed microcirculation does not increase the number of apoptotic cells at the ulcer edge in patients with venous disease. The reduced staining pattern with Bcl-2 at the ulcer edge seems not to result in higher susceptibility to apoptosis, but it remains to be proven whether it is involved in epidermal acanthosis.

Lubricating agents differ in their protection of cultured human epithelial cells against desiccation.

BACKGROUND: In dry eye, as a disease of the ocular surface, the instillation of artificial tears should compensate for the deficit in wetting and protect the mucosa against drying. MATERIAL/METHODS: The desiccation protection of different pharmacological substances was tested using the conjunctival epithelial cell line Chang 1-5c-4 (series 1) and the corneal cell line 2.040 pRSV-T (series 2). On confluent cell growth the cultures were wetted for 20 min with various preservative-free preparations of artificial tears The cell cultures were exposed to a constant air flow for 0, 15, 30 and 45 minutes. Cells were incubated with the vital dye Alamar Blue and subsequently absorption of the oxidised form of the dye was measured using an ELISA-Reader. RESULTS: Cell survival rates in series 1 after 0, 15, 30, 45 min were (1.02;0.81;0.35;0.32) for Artelac EDO, (0.82;0.69; 0.63;0.54) for Vidisic EDO, (0.77;0.80;0.67;0.70) for Vidisic Fluid EDO, (0.76;0.70;0.36; 0.34) for Acuolens, (0.97;0.46;0.35;0.33) for Viscofresh, (0.88;0.85;0.37; 0.33) for Hyal Drops SDU, (0.71;0.44;0.34;0.33) for PBS and in series 2 (1.03;0.84;-0.21;-0.20) for Artelac EDO, (0.89;0.92;0.93;0.86) for Vidisic EDO, (0.96;0.88;0.85;0.85) for Vidisic Fluid EDO, (1.01;0.75;-0.02;-0.03) for Acuolens, (0.98;0.17;-0.22;-0.20) for Viscofresh, (0.97;0.83;0.03;-0.21) for Hyal Drops SDU and (0.96;0.26;-0.24;-0.21) for PBS. Vidisic Fluid EDO and Vidisic EDO showed a significantly better protective effect after a drying period of 30 and 45 min. CONCLUSIONS: The protection capability of pharmacological substances against desiccation can be studied in a standardised cell culture system of human epithelial cell lines. Whether these in vitro results are conferrable to the efficacy of artificial tear drops in vivo has to be evaluated in clinical trials.

Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer.

Prostate cancer (PCA) is the most common type of cancer found in men of western countries and is the leading cancer death next to lung cancer and colorectal cancer. Prostate-specific antigen (PSA) test is an established diagnostic tool for PCA detection, but confirmation of diagnosis by histopathological evaluation of prostate needle biopsies is performed. To define protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH, n=11) and prostate cancer (PCA, n=12) patients by two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins were identified. The important proteins identified included prostatic acid phosphatase precursor, a significant overexpressed protein in PCA, prohibitin, NDRG1 tumor suppressor proteins, heat shock proteins, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH (n=13), moderate staining in prostate intra-epithelial neoplasia (PIN, n=5) but strong staining in PCA (n=18). Our results demonstrate that protein profiling and mRNA studies can be performed on the same prostate biopsy. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCA and BPH. Some of the interesting proteins identified in this approach may serve to develop new targets for PCA diagnosis and treatment.

MDR1 gene polymorphisms and risk of gingival hyperplasia induced by calcium antagonists.

BACKGROUND: Gingival overgrowth is a common side effect of calcium antagonists. Although the pathogenesis is unknown, several lines of evidence point to a modulation of inflammatory processes. Because the calcium antagonists, albeit to a variable degree, act as inhibitors of P-glycoprotein (P-gp), the gene product of multidrug resistance 1 (MDR1), and inflammation may modify P-gp expression, we analyzed the MDR1 polymorphisms as risk factors for gingival overgrowth induced by calcium antagonists. METHODS: Clinical, laboratory, and anamnestic data including periodontal parameters and use of calcium antagonists were assessed in a cross-sectional epidemiologic investigation (N = 1484). MDR1 polymorphisms in exon 21 G2677T/A and exon 26 C3435T were determined. P-gp expression was detected in gingival tissues. In a matched-pair analysis, 93 subjects using calcium antagonists and 186 not using them were compared. RESULTS: P-gp is expressed in the endothelial layers of blood vessels obtained from healthy or inflamed gingiva. Subjects treated with calcium antagonists had significantly deeper gingival pockets than their drug-free counterparts (P <.0001). This drug-related side effect was associated with the MDR1 2677G/G or G/TA genotype (P <.001) but not with the variant genotype T/TA. This drug effect was proved by multiple regression analysis with adjustment for the risk factors of periodontitis (age, sex, smoking, and education) (P <.0001) and was associated with elevated C-reactive protein levels. The association of probing depth with the MDR1 polymorphism was confirmed in the matched-pair analysis (P <.0001). CONCLUSION: Treatment with calcium antagonists leads to gingival hyperplasia, which is associated with the MDR1 G2677T/A polymorphism. The MDR1 genotype may modify the inflammatory response to the drugs.

Age-related differential expression of apoptosis-related genes in conjunctival epithelial cells.

PURPOSE: To investigate whether the expression of apoptosis-related genes in normal conjunctival epithelial cells is age-related (as a prerequisite to assessing whether dysregulation of apoptosis may be involved during degenerative diseases). METHODS: Differential expression of apoptosis-related genes (e.g. apoptosis protease-activating factor 1 [Apaf-1]; caspases [casp] 3, 5, 8 and 9; Bad, Bax, Bcl-2, Bim, c-myc, Bag-1, as well as p53) was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Samples were obtained from impression cytology (IC) specimens taken from 50 healthy subjects. Group A comprised 27 subjects aged 19-32 years and group B included 23 subjects aged 53-84 years. RESULTS: Reverse transcription-PCR revealed the detection of apoptosis-related m-RNAs as follows (group A compared to group B): Apaf-1 0%/0%; Bcl-2 0%/35%; Bim 0%/9%; Bag-1 0%/9%; p53 0%/4%; casp-3 11%/52%; casp-5 59%/48%; casp-8 44%/22%; casp-9 4%/9%; Bax 81%/52%; Bad 96%/56%, and c-myc 89%/96%. CONCLUSION: The data show an age-related expression of apoptosis-related genes such as casp-3, Bad, Bax and Bcl-2 in normal conjunctival cells. These results provide basic information which will help us understand the expression pattern of apoptotic genes during physiological ageing of the conjunctiva and the possible dysregulation of apoptotic genes during acute and chronic diseases such as dry eye disease, allergic conjunctivitis or cicatrizing conjunctivitis.

Immunolocalization of glycodelin in the genital tract of rats.

Glycodelin, also known as placental protein 14 has been predominantly localized to organs of the human genital tract. Unfortunately the physiological role of glycodelin is largely unknown since it depends on limited availability of tissues. Therefore, a suitable animal model to study the role of glycodelin would be desirable. Previously, it was shown that glycodelin mRNA is expressed in the genital tract of male and female rats. In the present study, we demonstrate the expression of glycodelin protein in male and female rats by immunohistochemistry and Western blot analysis. For this purpose a polyclonal antibody was generated against glycodelin peptide. In female rats, glycodelin was found in the epithelial gland cells of the uterus, epithelial cells of the fallopian tube as well as in corpora lutea, interstitial and theca cells of the ovary. Glycodelin was distributed in all epithelial cells of the epididymis and the seminal vesicle. In the seminiferous epithelium, glycodelin was seen in all developmental stages of spermatogonia and spermatocytes and in Sertoli cells. Whereas in the rat male reproductive tract glycodelin expression is slightly different from human or primate tissues, in organs of the rat female genital tract glycodelin expression is similar to humans and primates.

Loss of heterozygosity at 12p13 and loss of p27KIP1 protein expression contribute to melanoma progression.

Little is known about the mechanisms causing p27KIP1 decrease in melanomas. Therefore, we performed loss of heterozygosity (LOH) analysis with polymerase chain reaction at seven different loci surrounding the p27KIP1/CDKN1B gene at 12p13 and direct DNA sequencing analysis of all exons. Furthermore, the immunohistochemical expression of p27KIP1 and Ki-67 was investigated. Only two mutations in the sequence of p27KIP1/CDKN1B were detected, but the number of tumours showing LOH at 12p13 increased significantly with the parameters of tumour progression (pT level, P=0.018; Breslow index, P=0.01; Clark level, P<0.001), with a more aggressive tumour growth (radial versus vertical growth, P=0.018) and tumour subtype (superficial spreading melanomas versus nodular melanomas versus metastases, P<0.001). p27KIP1 protein expression decreased with the Clark level ( P=0.026) and the pT level ( P=0.045). No correlation between LOH affecting 12p13 and p27KIP1 protein decrease in melanomas was stated. This does not exclude the participation of p27KIP1/CDKN1B in p27KIP1 protein decrease, since protein expression is regulated at various cellular levels; but it could also suggest that other tumour suppressors are situated in the same region as p27KIP1/CDKN1B. Taken together, our data shows that loss of p27KIP1 protein expression and LOH at 12p13 contribute to tumour progression in melanoma.

Increased expression and altered location of annexin IV in renal clear cell carcinoma: a possible role in tumour dissemination.

The proteome of renal cell carcinoma and non-neoplastic kidney tissue was analysed from 12 patients by two-dimensional polyacrylamide gel electrophoresis to search for differentially expressed proteins in the tumour. Annexin IV was identified to be up-regulated in tumour cells. These patients and further 11 were characterized by RT-PCR. We found an increased amount of annexin IV mRNA. Immunohistochemical analysis revealed an altered localization of annexin IV in tumour cells. Additionally we demonstrate that over-expressed annexin IV promotes cell migration in a carcinoma model system. From these results above it seems possible that annexin IV plays an important role in the morphological diversification and dissemination of the clear cell renal cell carcinoma.

Human agmatinase is diminished in the clear cell type of renal cell carcinoma.

The proteome of RCC was analyzed by 2D PAGE to search for tumor-associated proteins. Agmatinase, which hydrolyzes agmatine to putrescine and urea, was identified by mass spectrometry and database searches and shown to be downregulated in tumor cells. Additionally, RT-PCR and Northern blot analyses demonstrated a clearly decreased amount of agmatinase mRNA in tumor cells. The differential expression of agmatinase mRNA was confirmed at the protein level. Western blot analysis showed almost no detectable agmatinase protein in tumor cells compared to corresponding normal renal tissue. Agmatinase mRNA is most abundant in human liver and kidney but expressed to a lesser extent in several other tissues, including skeletal muscle and small intestine. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Using transfection and immunohistochemical studies, we show that agmatinase is localized in the mitochondria. Immunohistochemical studies revealed that agmatinase in the normal kidney is restricted to tubulus epithelial cells, while in tumors staining was low and heterogeneous. Thus, expression of human agmatinase is altered in RCC. We discuss the consequences of these findings in terms of polyamine, NO metabolism and macrophage function.

Expression of AP-2alpha, c-kit, and cleaved caspase-6 and -3 in naevi and malignant melanomas of the skin. A possible role for caspases in melanoma progression?

Progression of melanoma is associated with loss of the transcription factor AP-2alpha and tyrosine-kinase receptor c-kit. However, the mechanisms by which these two proteins are down-regulated have not been fully elucidated. Fifty non-selected melanomas comprising ten superficial spreading melanomas (five exhibiting a radial growth phase and five a vertical growth phase), ten primary nodular melanomas, 30 melanoma metastases, and 16 naevi were investigated by direct sequencing analysis of the AP-2alpha and c-kit genes and by immunohistochemistry for the respective proteins. Because it has recently been demonstrated that AP-2alpha is preferentially cleaved by caspase-6 and to a lesser extent by caspase-3, immunohistochemistry for the cleaved (activated) forms of caspase-6 (c-casp-6) and caspase-3 (c-casp-3) was carried out. No mutations were identified in the c-kit gene, but three different point mutations were demonstrated in the activation motif of AP-2alpha in four tumours: one vertical growth phase superficial spreading melanoma, one nodular melanoma, and two metastases. Immunohistochemistry revealed progressive loss of the AP-2alpha and c-kit proteins in primary melanomas and metastases when compared with naevi. The decrease of both markers was more accentuated in the dermal component of all primary tumours, with c-kit more affected than AP-2alpha. All invasive melanomas and metastases expressed c-casp-6. c-casp-3 was expressed by 83% of the metastases and in the dermal component of one nodular melanoma. These findings suggest that the loss of AP-2alpha protein expression during the progression of melanoma could be related to mutation of the gene in only a small number of tumours, whereas the expression and activation of caspases, most prominently caspase-6, may be an important factor for the down-regulation of AP-2alpha protein. Furthermore, this study supports recent data that the activation of caspases does not inevitably result in apoptosis, but may also contribute to tumour progression in melanomas.