Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector.

Although the monomeric GTPases RalA and RalB have been shown to regulate a variety of transcription factors, little is known regarding the differences or similarities in transcriptional programs regulated by RalA compared to RalB. Further, the association of these transcriptional pathways to human carcinogenesis and progression remains unclear. Here, we studied the role of RalA and/or RalB in transcriptional regulation by combining short interfering RNA depletion of Ral with gene expression profiling via microarray in the human bladder cancer cell line, UMUC-3. A large number of genes were found to be similarly modulated in cells with RalA and RalB depletion, suggesting that RalA and RalB impinge on overlapping transcriptional signaling pathways. However, smaller sets of genes were modulated by depletion of RalA or RalB, indicating that these closely related proteins also regulate nonoverlapping transcriptional pathways. Computational analysis of upstream sequences of genes modulated by Ral depletion identified Ras-responsive element-binding protein (RREB)-1, as a putative Ral transcriptional target, which we verified experimentally. Importantly, as a group, Ral-regulated probe sets identified here were disproportionally represented among those differentially expressed as a function of human bladder transformation. Taken together, these data strongly suggest that Ral family members mediate both common and specific transcriptional programs that are associated with human cancer and identify RREB-1 as a novel transcriptional effector of Ral.

Spatial variation in colour morph, spotting and allozyme frequencies in the candy-stripe spider, Enoplognatha ovata (Theridiidae) on two Swedish archipelagos.

The selective significance, if any, of many invertebrate visible polymorphisms is still not fully understood. Here we examine colour- and black spotting-morph frequencies in the spider Enoplognatha ovata in populations on two Swedish archipelagos with respect to different spatial scales and, in one archipelago, against the background of variation at four putative neutral allozyme marker loci. Every population studied was polymorphic for colour and 28 out of 30 contained all three colour morphs--lineata, redimita and ovata. We found no evidence for a breakdown in the traditional colour morph designation previously suggested for other northern European populations of this species. For colour there is no significant heterogeneity at spatial scales greater than between local populations within islands. Black spotting frequencies show a similar lack of pattern over larger spatial scales except that there are significant differences between the Stockholm and Göteborg archipelagos. Measures of population differentiation (theta) within the Stockholm islands for the two visible systems show them to be significantly more differentiated than the neutral markers, suggesting local selection acting on them in a population-specific manner. On the basis of previous observations and the distribution of spotting phenotypes on a European scale, it is argued that thermal selection might operate on black spotting during the juvenile stages favouring more spots in continental climates. It is not clear what selective forces act on colour.

Genetic drift within a protected polymorphism: enigmatic variation in color-morph frequencies in the candy-stripe spider, Enoplognatha ovata.

The candy-stripe spider, Enoplognatha ovata, exhibits a striking color polymorphism comprising three morphs. A number of lines of evidence strongly suggest that this polymorphism is maintained by natural selection: its presence in a sister species, E. latimana; the physical nature of the variation; the virtual lack of monomorphic populations; the highly consistent rank-order of morphs within populations; and the presence of large-scale clines associated with climatic variables. However, the absence of selection is equally strongly suggested by very local surveys of morph frequencies over space and time, perturbation experiments, and a variance in morph frequency between populations that is virtually independent of spatial scale. In addition, local spatial patterns in one study site (Nidderdale, Yorkshire, England) have been explained in terms of intermittent drift over half a century ago, a hypothesis supported here by the distributions of four other genetic markers (two allozyme and two visible polymorphisms). A heuristic model is suggested that reconciles these apparently contradictory messages regarding the importance of drift and selection in this system. It is proposed that when allele frequencies of the color morph redimita lie between approximately 0.05 and 0.3, the deltaq on q plot is very shallow, so that within this region, where the majority of populations lie, selection is weak and drift is the major force determining local morph frequencies. However, outside this range of frequencies, powerful selection acts to protect the polymorphism. This model may apply to polymorphisms in other species and explain why evidence of selection in natural populations is often elusive.

Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer.

PURPOSE: RhoGDI2 was recently shown to be a metastasis suppressor gene in models of bladder cancer. We sought to further understand its importance in human cancer by determining the level of its expression and the distribution of its encoded protein in normal human tissues and cell lines and to evaluate whether its protein expression is a determinant of human bladder cancer progression. EXPERIMENTAL DESIGN: RhoGDI2 mRNA and protein expression was evaluated in cell lines and human tissues using Affymetrix and tissue microarrays, respectively. Tissue microarrays represented most human normal adult tissues and material from 51 patients that had undergone radical cystectomy for bladder cancer. In these 51 patients, the chi(2) test was used to test for associations between RhoGDI2 and stage, grade of urothelial carcinoma, histological type, and disease-specific survival status. Cox proportional hazards regression analyses were used to estimate the effect of RhoGDI2 expression level on time to development of metastatic disease and disease-specific survival time, adjusting for grade, stage, and histological type. RESULTS: In normal tissues, there was strong RhoGDI2 protein expression in WBCs, endothelial cells, and transitional epithelium. RhoGDI2 mRNA expression was inversely related to the invasive and metastatic phenotype in human bladder cancer cell lines. In patients with bladder cancer, univariate analysis indicated that reduced tumor RhoGDI2 protein expression was associated with a lower actuarial 5-year disease-free and disease-specific survival (P = 0.01). In addition, patients with tumors that had low or absent RhoGDI2 had a shorter time to disease-specific death (P

Nicotine inhibits voltage-dependent sodium channels and sensitizes vanilloid receptors.

Nicotine is an alkaloid that is used by large numbers of people. When taken into the body, it produces a myriad of physiological actions that occur primarily through the activation of neuronal nicotinic acetylcholine receptors (nAChRs). We have explored its ability to modulate TRPV1 receptors and voltage-gated sodium channels. The reason for investigating nicotine's effect on sodium channels is to obtain a better understanding of its anti-nociceptive properties. The reasons for investigating its effects on capsaicin-activated TRPV1 channels are to understand how it may modulate this channel that is involved in pain, inflammation, and gustatory physiology. Whole cell patch-clamp recordings from rat trigeminal ganglion (TG) nociceptors revealed that nicotine exhibited anesthetic properties by decreasing the number of evoked action potentials and by inhibiting tetrodotoxin-resistant sodium currents. This anesthetic property can be produced without the necessity of activating nAChRs. Nicotine also modulates TRPV1 receptors inducing a several-fold increase in capsaicin-activated currents in both TG neurons and in cells with heterologously expressed TRPV1 receptors. This sensitizing effect does not require the activation of nAChRs. Nicotine did not alter the threshold temperature (approximately 41 degrees C) of heat-activated currents in TG neurons that were attributed to arise from the activation of TRPV1 receptors. In this regard, its effect on TRPV1 receptors differs from those of ethanol that has been shown to increase the capsaicin-activated current but decrease the threshold temperature. These studies document several new effects of nicotine on channels involved in nociception and indicate how they may impact physiological processes involving pain and gustation.

Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation.

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [(3)H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [(3)H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

Critical issues in PhD training for biomedical scientists.

The rapidly changing world of modern biomedical research is raising important new issues for traditional PhD training programs and is creating concern among young PhD scientists about their futures. Specifically, the United States is producing substantially more biomedical PhDs than can be accommodated in professional positions that truly require the PhD as a credential. The "surplus" PhD population is being relegated to poorly paid, unstable, and increasingly unsatisfying jobs. In addition, many current graduate and postdoctoral training programs may not be adequately preparing young scientists for the more complex, more quantitative biological science of the future. Finally, many current graduate training programs are not attracting a sufficient portion of the most talented young people in the nation. To ameliorate these problems in the training and early career paths of basic biomedical scientists, the authors make specific recommendations, such as urging (1) that graduate trainees should be supported exclusively by competitive individual fellowships, training grants, or institutional funds and not by RO1s or similar research awards; (2) that graduate and postdoctoral stipends be increased so that they provide a reasonable living wage; and (3) that research-intensive academic institutions create a career path for biomedical PhDs other than that designed for the traditional tenure-track, grant-funded principal investigator and faculty member. They conclude that it is in the interest of faculty and institutions to make these and other drastic changes because the current system is both inherently unfair and self-destructive.

Reduced oral wound healing in the NOD mouse model for type 1 autoimmune diabetes and its reversal by epidermal growth factor supplementation.

Using the NOD mouse, a model for type 1 diabetes, we examined how reduced concentrations of epidermal growth factor (EGF) in the saliva, after onset of type 1 diabetes, affect oral wound healing. Diabetic NOD/LtJ mice on insulin therapy, prediabetic NOD/LtJ, and age- and sex-matched BALB/cJ mice were given a cutaneous tongue punch and allowed to undergo normal healing. With diabetes onset and a reduction in saliva-derived growth factor levels, the rate of tongue wound healing was reduced compared with nondiabetic NOD/LtJ and healthy BALB/cJ mice. Addition of exogenous EGF to the drinking water did not accelerate the rate of healing in BALB/cJ or prediabetic NOD/LtJ; however, diabetic NOD/LtJ mice exhibited accelerated wound healing similar to healthy mice. These results demonstrate that loss of growth factors from saliva is associated with profoundly reduced oral wound healing, suggesting that therapeutic treatment with topical delivery may be beneficial to patients with type 1 diabetes and oral wound complications.

Differential absorption and distribution of epidermal growth factor and insulin-like growth factor in diabetic NOD mice.

Previous studies have shown that absorption of growth factors occurs through the gastrointestinal tract and the oral cavity. The non-obese diabetic (NOD) mouse, a model for spontaneous development of type 1 insulin-dependent diabetes (IDDM), was evaluated for the absorption and systemic distribution of growth factors. Radiolabeled epidermal growth factor (EGF) and insulin-like growth factor, type I (IGF-I), were administered by gavage into the stomach or by lozenge into the sublingual vasculature of either diabetic or nondiabetic mice. After a time-dependent uptake, the levels of absorption and distribution through the tissues were measured. A similar time course of EGF absorption following gavage administration was determined for NOD and C57BL/6 mice, with a maximum tissue distribution by 30-min post infusion. Diabetic NOD mice showed similar levels of IGF uptake and tissue distribution compared with nondiabetic NOD and normal healthy C57BL/6 mice, whether administered by gavage or sublingual lozenge. On the other hand, gavage uptake and tissue distribution of EGF was significantly higher in diabetic mice when compared to sublingual administration in nondiabetic NOD or C57BL/6 healthy control mice. These findings suggest that the overall potential uptake and distribution of saliva-derived growth factors in systemic wound-healing processes is retained with diabetes onset, and may offer a new avenue to treating this complication of diabetes.

Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate-dependent epithelial chloride channel.

BACKGROUND & AIMS: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. METHODS: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. RESULTS: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. CONCLUSIONS: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.

Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation.

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.

Salivary EGF levels reduced in diabetic patients.

Oral problems such as periodontitis are recognized major complications associated with diabetes. Salivary derived growth factors, including epidermal growth factor (EGF), are thought to play a role in helping maintain levels of oral health, promoting wound healing, and maintaining mucosal integrity. In the present study, salivary levels of EGF in diabetic vs. healthy control patients was evaluated. Twenty-one diabetic patients participated in this study. Age, race, sex and smoking histories were matched with 21 systematically healthy nondiabetic patients. Three milliliters of unstimulated resting whole saliva was collected from each patient at 6 h intervals up to 42 h and whole saliva protein concentrations were determined for each sample. EGF concentrations for each sample were quantitated spectrophometrically utilizing an immunoassay. Diabetic patients had greater salivary protein concentrations over 42 h of collection with a mean of 1.502+/-0.09 vs. 1.242+/-0.05 mg/ml for healthy control patients. The EGF concentration was significantly lower (p<0.05) for the diabetic patients compared to control patients, whether expressed relative to 1 ml volume of saliva (873.43+/-106.5 vs. 1101.09+/-116.8 pg/ml) or 1 mg whole saliva protein (629.18+/-92.6 vs. 931.20+/-124.6 pg/mg saliva protein). This study suggests that reduced levels of salivary EGF in diabetic patients may contribute to the development of oral and systemic complications of diabetes, which may have future clinical applications.

Dominant-negative mutants identify a role for GIRK channels in D3 dopamine receptor-mediated regulation of spontaneous secretory activity.

The human D3 dopamine receptor can activate G-protein-coupled inward rectifier potassium channels (GIRKs), inhibit P/Q-type calcium channels, and inhibit spontaneous secretory activity in AtT-20 neuroendocrine cells (Kuzhikandathil, E.V., W. Yu, and G.S. Oxford. 1998. Mol. Cell. Neurosci. 12:390-402; Kuzhikandathil, E.V., and G.S. Oxford. 1999. J. Neurosci. 19:1698-1707). In this study, we evaluate the role of GIRKs in the D3 receptor-mediated inhibition of secretory activity in AtT-20 cells. The absence of selective blockers for GIRKs has precluded a direct test of the hypothesis that they play an important role in inhibiting secretory activity. However, the tetrameric structure of these channels provides a means of disrupting endogenous GIRK function using a dominant negative approach. To develop a dominant-negative GIRK mutant, the K(+) selectivity amino acid sequence -GYG- in the putative pore domain of the human GIRK2 channels was mutated to -AAA-, -GLG-, or -GFG-. While the mutation of -GYG- to -GFG- did not affect channel function, both the -AAA- and -GLG- GIRK2 mutants were nonfunctional. This suggests that the aromatic ring of the tyrosine residue rather than its hydroxyl group is involved in maintaining the pore architecture of human GIRK2 channels. When expressed in AtT-20 cells, the nonfunctional AAA-GIRK2 and GLG-GIRK2 acted as effective dominant-negative mutants and significantly attenuated endogenous GIRK currents. Furthermore, these dominant-negative mutants interfered with the D3 receptor-mediated inhibition of secretion in AtT-20 cells, suggesting they are centrally involved in the signaling pathway of this secretory response. These results indicate that dominant-negative GIRK mutants are effective molecular tools to examine the role of GIRK channels in vivo.

Aberrant proteolytic digestion of biglycan and decorin by saliva and exocrine gland lysates from the NOD mouse model for autoimmune exocrinopathy.

OBJECTIVE: The protein components of the extracellular matrix (ECM) are responsible for driving tissue morphogenesis, the development of differentiated function, and the sequestration of biologically active molecules such as growth factors in close proximity to tissue and organ cells. Recent reports indicate that saliva and exocrine tissue lysates from Sjögren's syndrome patients and the non-obese diabetic (NOD) mouse model for autoimmune exocrinopathy demonstrate elevated levels of specific enzymes that degrade the ECM, especially the matrix metalloproteinases (MMPs). To determine if elevated levels of MMPs could be important in exocrine tissue destruction, we examined proteolytic activity against two ECM proteoglycans, decorin and biglycan. METHODS: Purified recombinant human core protein for decorin or biglycan was incubated with saliva or gland lysates from either control BALB/c or NOD mice. Degraded proteoglycan products were estimated by Western blotting analysis using anti-decorin or anti-biglycan monospecific polyclonal antibodies. The levels of TGF beta protein were measured by ELISA. RESULTS: Proteolytic activity for decorin and biglycan was not observed in the saliva and salivary gland lysates collected from C57BL/6 or BALB/c mice used as normal controls. In contrast, both proteoglycans were degraded by saliva and exocrine gland lysates from NOD mice and the congenic partner strains NOD-scid and NOD.B10.H2b. This proteolytic activity for proteoglycans was inhibited by the MMP inhibitors, EDTA, GM6001 and 1,10-phenanthroline. Protein steady state levels for TGF beta were increased in the saliva and gland lysates from 20-week old NOD strains, as compared to BALB/c mice and NOD treated with the MMP inhibitor GM6001. With the inhibition of MMP activity, TGF beta levels declined in saliva and gland lysates. CONCLUSION: Proteolytic degradation of the ECM molecules decorin and biglycan is elevated in the exocrine tissues of the NOD mouse model for Sjögren's syndrome. Furthermore, the proteolysis of small leucine-rich proteoglycans correlates with the presence of elevated levels of TGF beta in gland lysates and saliva.

Regulation of cell movement is mediated by stretch-activated calcium channels.

Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeletons, and the formation and disassembly of cell-substratum adhesions. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily 'stuck' to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein.

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.

Activation of human D3 dopamine receptor inhibits P/Q-type calcium channels and secretory activity in AtT-20 cells.

The D3 dopamine receptor is postulated to play an important role in the regulation of neurotransmitter secretion at both pre- and postsynaptic terminals. However, this hypothesis and the underlying mechanisms remain untested because of the lack of D3-selective ligands, paucity of appropriate model secretory systems, and the weak and inconsistent coupling of D3 receptors to classical signal transduction pathways. The absence of ligands that selectively discriminate between D3 and D2 receptors in vivo precludes the study of D3 receptor function in the brain and necessitates the use of heterologous expression systems. In this report we demonstrate that activation of the human D3 dopamine receptor expressed in the AtT-20 neuroendocrine cell line causes robust inhibition of P/Q-type calcium channels via pertussis toxin-sensitive G-proteins. In addition, using the vesicle trafficking dye FM1-43, we show that D3 receptor activation significantly inhibits spontaneous secretory activity in these cells. Our results not only support the hypothesis that the D3 receptor can regulate secretory activity but also provide insight into the underlying signaling mechanisms. We propose a functional model in which the D3 receptor tightly regulates neurotransmitter release at a synapse by only allowing the propagation of spikes above a certain frequency or burst-duration threshold.

Elevated levels of human salivary epidermal growth factor after oral and juxtaoral surgery.

PURPOSE: Saliva provides a natural reservoir of growth factors whose purposes have remained elusive. Animal studies suggest that saliva-derived growth factors play a role in systemic and oral wound healing. In the current study, salivary concentrations of epidermal growth factor (EGF) were monitored in patients before and after oral and juxtaoral surgery. PATIENTS AND METHODS: Whole resting saliva was collected from a group of patients with parotid gland tumors requiring surgical resection. Another group of patients a history of periodontal disease requiring surgical intervention also provided whole salivary samples. Healthy age- and sex-matched persons served as controls. RESULTS: Salivary EGF levels were elevated in both groups of patients within 24 hours after surgery. In the periodontitis patients, a second smaller peak was assayed noted between 36 and 48 hours. After this, EGF concentrations returned to levels comparable to healthy controls in both experimental groups. CONCLUSIONS: Although the local cells have the ability to synthesize and secrete growth factors at a site of injury, these results suggest that surgery stimulates increased synthesis and secretion of growth factors in the saliva as well. This increased level of saliva-derived growth factor may also aid in promoting wound healing.

Excessive synthesis of matrix metalloproteinases in exocrine tissues of NOD mouse models for Sjögren's syndrome.

OBJECTIVE: Matrix metalloproteinases (MMP) and their substrates, components of the extracellular matrix, regulate environmental signals for cellular differentiation and tissue function. Changes in the levels of these enzymes may influence cell survival as well as pathology involving ectopic apoptosis. Using the non-obese diabetic (NOD) mouse model for Sjögren's syndrome, we evaluated the synthesis and expression of MMP in the exocrine target tissues of autoimmunity. METHODS: NOD, immunodeficient NOD-scid, and nondiabetic NOD.B10.H2b mice were evaluated for MMP activity in their saliva and exocrine gland lysates by gelatin zymography and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, changes in protein content of saliva and gland lysates were determined by specific Western blot and by enzymatic activity of amylase and cysteine proteases. Mice continuously treated with the MMP inhibitor GM6001 were evaluated from 7 to 20 weeks of age for the contribution of MMP activity to development of these hallmark biochemical markers of Sjogren's syndrome-like disease of NOD mice. RESULTS: Gelatin zymography of whole saliva and gland lysates indicated the presence of increased proteolytic activity, corresponding to proteins with a molecular mass ranging from 50 to 95 kDa, in the saliva of older (> 20 weeks of age) NOD mice as well as NOD.B10.H2b and NOD-scid mice compared to BALB/c controls. Elevated steady state levels of mRNA transcripts for the gelatinases MMP-2 and MMP-9 were detected in total RNA extracted from parotid and submandibular glands by RT-PCR. Despite prophylactic injection of the broad spectrum MMP inhibitor GM6001 into mice beginning at 7 weeks of age and continuing to 20 weeks, development of the autoimmune exocrinopathy was neither stopped nor retarded. CONCLUSION: These observations suggest that excessive MMP activity is associated with autoimmune Sjögren's syndrome-like disease in NOD mice. However, a possible contribution by increased MMP activity in initiation and progression of this autoimmune disease is yet to be elucidated.