Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar.

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmon Salmo salar. Emphasis was placed on Na(+) , K(+) -ATPase (NKA) and Na(+) , K(+) , Cl(-) co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na(+) , Cl(-) co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Osmoregulation in Atlantic salmon Salmo salar smolts transferred to seawater at different temperatures.

In order to investigate how changes in gill Na(+) , K(+) -ATPase (NKA) α1a and α1b subunits, Na(+) , K(+) , 2Cl(-) co-transporter (NKCC1) and the apical cystic fibrosis trans-membrane conductance regulator-I (CFTR-I) transcripts in wild strain of Atlantic salmon, Salmo salar, smolts are affected by temperature during spring, hatchery-reared parr (mean ± s.e. fork length = 14·1 ± 0·5; mean ± s.e. body mass = 28·5 ± 4·5 g) originating from broodstock from the Vosso river (western Norway) were acclimated to three temperature regimes (4·1, 8·1 and 12·9° C) in May and reared under gradually increasing salinity between May and June. Changes in plasma Na(+) , haematocrit (Hct) and PCO2 were monitored in order to assess and compare key physiological changes with the transcriptional changes in key ion transporters. The temperatures reflect the natural temperature range in the River Vosso during late spring. Overall, higher gill NKA α1b mRNA levels, gill NKCC1a levels and CFTR-I levels were observed in the 4·1° C group compared to the 11·9° C group. This coincided with a 2-3 week period with decreased Hct and PCO2 and may indicate a critical window when smolts suffer from reduced physical performance during migration. Further research is needed to confirm the potential interaction between ecological and physiological conditions on mortality of hatchery-reared smolts from River Vosso during their natural migration.

Plasma growth hormone-binding protein levels in Atlantic salmon Salmo salar during smoltification and seawater transfer.

Specific growth hormone (GH)-binding protein (Ghbp) was purified from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss plasma with immunoprecipitation and characterized in cross-linking studies using autoradiography and western blots. The size of the Ghbp was estimated to be c. 53 kDa. A radioimmunoassay was established to measure Ghbp in salmonids, using antibodies specific against the extracellular segment of the S. salar growth hormone receptor 1 (grh1; GenBank AY462105). Plasma Ghbp levels were measured in S. salar smolts in fresh water and after transfer to seawater (SW; experiments 1 and 2), and in post-smolts kept at different salinities (0, 12, 22 and 34) for 3 months (experiment 3). A transient increase in plasma Ghbp, which lasted for 1 month or less, was noted in smolts after transfer to SW. Concomitantly, plasma GH and gill Na(+) -K(+) -ATPase activity increased during smoltification (in experiment 2). No difference in plasma Ghbp was evident between post-smolts kept at different salinities, although the fish kept at salinity 34 had higher plasma GH than the group kept at salinity 22 and higher hepatic ghr1 expression than post-smolts kept at salinity 12. This suggests that plasma Ghbp regulation may respond to salinity changes in the short term. The lack of correlation between Ghbp, plasma GH and hepatic ghr1 expression in the long-term post-smolt experiment indicates that Ghbp levels may be regulated independently of other components of the endocrine GH system in salmonids.

Aluminum exposure impacts brain plasticity and behavior in Atlantic salmon (Salmo salar).

Aluminum (Al) toxicity occurs frequently in natural aquatic ecosystems as a result of acid deposition and natural weathering processes. Detrimental effects of Al toxicity on aquatic organisms are well known and can have consequences for survival. Fish exposed to Al in low pH waters will experience physiological and neuroendocrine changes that disrupt homeostasis and alter behavior. To investigate the effects of Al exposure on both the brain and behavior, Atlantic salmon (Salmo salar) kept in water treated with Al (pH 5.7, 0.37±0.04 µmol 1(-1) Al) for 2 weeks were compared with fish kept in under control conditions (pH 6.7, <0.04 µmol 1(-1) Al). Fish exposed to Al and acidic conditions had increased Al accumulation in the gills and decreased gill Na(+), K(+)-ATPase activity, which impaired osmoregulatory capacity and caused physiological stress, indicated by elevated plasma cortisol and glucose levels. Here we show for the first time that exposure to Al in acidic conditions also impaired learning performance in a maze task. Al toxicity also reduced the expression of NeuroD1 transcript levels in the forebrain of exposed fish. As in mammals, these data show that exposure to chronic stress, such as acidified Al, can reduce neural plasticity during behavioral challenges in salmon, and may impair the ability to cope with new environments.

Physiology during smoltification in Atlantic salmon: effect of melatonin implants.

Melatonin implants were used to override natural melatonin rhythm in groups of juvenile Atlantic salmon, Salmo salar, raised at simulated natural photoperiod (SNP) and constant light (LL) from mid-March until end of August. The experiment contained also both sham control (with non-melatonin implants) and control (no implants). No differences were found in the experimental variables between these two control groups. Growth and food intake were negatively affected by melatonin implantation. Overall, higher GH levels were observed in the SNP melatonin-implanted group, whereas no differences in GH levels were seen between the SNP control, LL control, or the LL melatonin-implanted groups. Highest food intake was seen in the LL control group. No differences in food intake were recorded between the LL melatonin-implanted and SNP control groups. Gill Na(+), K(+), ATPase (NKA) activity was influenced by time as well as the interaction between photoperiod and time. No differences in gill NKA activity or plasma chloride levels following transfer to seawater were seen between the groups with melatonin implants and their controls. Based on the present results, it seems apparent that melatonin does play a role in regulating food intake and growth in Atlantic salmon smolts.

Seawater tolerance and post-smolt migration of wild Atlantic salmon Salmo salar × brown trout S. trutta hybrid smolts.

High levels of hybridization between Atlantic salmon Salmo salar and brown trout Salmo trutta have been reported in the River Driva. This study presents the underlying mechanisms of development of seawater (SW) tolerance and marine migration pattern for S. salar×S. trutta hybrids. Migrating S. salar×S. trutta hybrid smolts caught in the River Driva, Norway (a river containing Gyrodactylus salaris), displayed freshwater (FW) gill Na(+), K(+) -ATPase (NKA) activity levels of 11·8 µmol ADP mg protein h(-1), which were equal to or higher than activity levels observed in S. salar and S. trutta smolts. Following 4 days of SW exposure (salinity 32·3), enzyme activity remained high and plasma ion levels were maintained within the normal physiological range observed in S. salar smolts, indicating no signs of ion perturbations in S. salar×S. trutta hybrids. SW exposure induced an increase in NKA α1b-subunit mRNA levels with a concurrent decrease in α1a levels. Salmo salar×S. trutta post-smolts migrated rapidly through the fjord system, with increasing speed with distance from the river, as is often seen in S. salar smolts. The present findings suggest that S. salar×S. trutta smolts, as judged by the activity and transcription of the NKA system, regulation of plasma ion levels and migration speed more closely resemble S. salar than S. trutta.

Long-term effects of photoperiod, temperature and their interaction on growth, gill Na⁺, K⁺-ATPase activity, seawater tolerance and plasma growth-hormone levels in Atlantic salmon Salmo salar.

This study was undertaken to examine the long-term effects of photoperiod, temperature and their interaction on growth, gill Na⁺,K⁺-ATPase (NKA) activity, seawater tolerance and plasma growth-hormone levels in Atlantic salmon Salmo salar pre-smolts and smolts. The fish (mean ± s.e. initial body mass = 15·9 ± 0·4 g) were reared on two photoperiods (continuous light, LL, and simulated natural photoperiod, LDN, 60° 25' N) and two temperatures (8·3 and 12·7° C) from June to May of the following year. Mean body mass was affected by photoperiod, temperature and their interactions. Both temperature groups on LL developed peak levels in gill NKA activity from October to November, 4-5 months prior to the natural season for the parr-smolt transformation. Fish at 12° C showed peak levels in NKA activity 4-6 weeks before the fish at 8° C. Fish in all four experimental groups showed maximum NKA activity within a similar size range (113-162 g). The present findings further indicate that smoltification in S. salar is to some extent driven by size, and that S. salar will develop smolt characteristics, e.g. a marked increase in NKA activity, within a similar size range. Faster-growing S. salar will, thus, reach this size threshold at a relatively younger age.

Hand radiology characteristics of patients carrying the T(303)M mutation in the gene for matrilin-3.

OBJECTIVE: To determine whether the recently described hand osteoarthritis (HOA)-associated T(303)M mutation in the gene for matrilin-3 (MATN3) is associated with specific radiological changes on hand radiographs. METHOD: Standard hand radiographs from 26 HOA patients carrying the T(303)M missense mutation in the MATN3 gene (T(303)M patients) were compared with those from 52 HOA controls matched for sex, age, and clinical disease severity. Two blinded readers scored the radiographs, using the Verbruggen-Veys anatomical scoring system for the interphalangeal and metacarpophalangeal joints and the OARSI atlas scoring system for the first carpometacarpal (CMC1) joints. A scoring system based on the latter was used for the scaphoid-trapezoid-trapezoideum (STT) joints. RESULTS: No particular distinguishing features were found in the T(303)M patients and the prevalence of erosive and cystic changes was similar to the control group. As a group, however, the T(303)M patients had more severe thumb-base affection, particularly in the STT joint. Thus, definite radiological OA in both CMC1 and STT joints and higher STT scores compared with CMC1 were significantly more common in patients carrying the T(303)M mutation. Radiological scores for joint-space narrowing (CMC1 and STT) and osteophytes (STT) were also significantly higher in the T(303)M patients. CONCLUSION: Patients carrying the T(303)M mutation in the gene for matrilin-3 express a form of HOA that is radiologically indistinguishable from idiopathic HOA in individual patients but they have more severe thumb-base involvement, particularly in the STT joint. This is the first described genetic mutation that is associated with a common form of osteoarthritis.

Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization.

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.

A large Icelandic family with early osteoarthritis of the hip associated with a susceptibility locus on chromosome 16p.

OBJECTIVE: To describe a large kinship with inherited hip osteoarthritis (OA) and its associated susceptibility locus. METHODS: Four generations of a kinship with familial hip OA were identified and characterized by family history and by clinical, radiographic, and histopathologic examination. In the genome-wide search for a susceptibility locus, OA cases were defined as those who had undergone total hip replacement associated with a clinical and radiographic diagnosis of hip OA. A genome-wide scan was performed using a framework set of microsatellite markers with an average spacing of 10 cM. RESULTS: The hip OA of this family was indistinguishable from that of idiopathic, nonfamilial hip OA. There was no apparent evidence of spondyloepiphyseal dysplasia or other dysplasias usually associated with mutations in collagen genes. The genome-wide scan revealed a locus on chromosome 16p between 28 cM and 47 cM from the telomere, and this locus met the criteria for suggestive linkage (multipoint allele-sharing logarithm of odds [LOD] score 2.58, P = 1.6 x 10(-4)). Two additional regions with LOD scores of >1.5 were obtained. CONCLUSION: We have identified and described the largest kinship with familial hip OA reported to date. Evidence for linkage in this family suggests that a gene for susceptibility to hip OA exists on chromosome 16p. This represents an independent identification of a susceptibility locus previously reported for hip OA in this geographic region.

Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.

Three forms of GnRH in the brain and pituitary of the turbot, Scophthalmus maximus: immunological characterization and seasonal variation.

Three forms of GnRH, chicken (c) GnRH-II, salmon (s) and seabream (sb) GnRH, were immunologically characterized in the brain and pituitary of turbot by ELISA. cGnRH-II and sGnRH were detected in the brain, while sbGnRH and sGnRH (but not cGnRH-II) were detected in the pituitary. In females, the levels of cGnRH-II in the turbot brain extracts increased from May to July, concomitant with an increase in oocyte diameter. In the pituitary, sbGnRH was found to be the dominant form, with levels 100-600-fold those of sGnRH. Both sGnRH and sbGnRH in the pituitary showed variation during the spawning season; sbGnRH increased from May to July and correlated with the increase in oocyte diameter, while sGnRH decreased. The overall patterns were the same for male turbot, although levels were generally lower. These findings suggest that sbGnRH could be controlling reproduction in the turbot. However, the seasonal variation in sGnRH indicates a potential physiological role in turbot reproduction. This study gives the first immunological indications that sbGnRH is present in the pituitary of a pleuronectiform fish, and will provide the basis for further studies on the endocrine regulation of reproduction in flatfish.

Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1.

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.

[Wada test. An investigation of language and memory functions in epileptic patients evaluated for temporal lobectomy.].

INTRODUCTION: Intracarotid sodium amytal injection was introduced as a clinical investigation of epileptic patients by Juhn Wada around 1950. The Wada test causes a brief inhibition of cerebral functions of the anaesthetized hemisphere, thus allowing tests to be performed on the contralateral hemisphere. The test is widely used to lateralize language functions and to assess the risk of postoperative amnesia in epileptic patients evaluated for temporal lobectomy Subjects and methods: Five epileptic patients were investigated. Three patients had hippocampal sclerosis and two had a benign tumour in the amygdala region. The sodium amytal was first injected to the hemisphere with seizure onset. After the development of paralysis of the contralateral side of the body, language and memory functions of the non-anaestetized hemisphere were assessed. The test was then repeated for the other hemisphere. RESULTS: The left hemisphere was dominant for language in three patients. In one patient the right hemisphere was dominant for language and in another patient language was bilaterally represented. In the three patients with hippocampal sclerosis, verbal and nonverbal memory was worse on the side of the lesion. This difference was not as marked for the two patients with lesion in the amygdala region. Total memory score was worse on the side of the lesion in all five patients. DISCUSSION: In both right and left handed individuals language is usually located in the left hemisphere. When epileptic seizures, with onset in the left hemisphere, start early in life, the language function can be transferred to the right hemisphere. This is a likely explanation for the right hemisphere language dominance in one patient. In all patients total memory score was lower for the hemisphere with seizure onset. This is in agreement with the suggestion of a lateralizing value of the Wada test.

Glycoprotein 330/low density lipoprotein receptor-related protein-2 mediates endocytosis of low density lipoproteins via interaction with apolipoprotein B100.

The ability of glycoprotein 330/low density lipoprotein receptor-related protein-2 (LRP-2) to function as a lipoprotein receptor was investigated using cultured mouse F9 teratocarcinoma cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which induces F9 cells to differentiate into endoderm-like cells, produced a 50-fold increase in the expression of LRP-2. Levels of the other members of the low density lipoprotein (LDL) receptor (LDLR) family, including LDLR, the very low density lipoprotein receptor, and LRP-1, were reduced. When LDL catabolism was examined in these cells, it was found that the treated cells endocytosed and degraded at 10-fold higher levels than untreated cells. The increased LDL uptake coincided with increased LRP-2 activity of the treated cells, as measured by uptake of both 125I-labeled monoclonal LRP-2 antibody and the LRP-2 ligand prourokinase. The ability of LDL to bind to LRP-2 was demonstrated by solid-phase binding assays. This binding was inhibitable by LRP-2 antibodies, receptor-associated protein (the antagonist of ligand binding for all members of the LDLR family), or antibodies to apoB100, the major apolipoprotein component of LDL. In cell assays, LRP-2 antibodies blocked the elevated 125I-LDL internalization and degradation observed in the retinoic acid/dibutyryl cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed that was likely mediated by LDLR since it could not be inhibited by LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated protein, or apoB100 antibody. The results indicate that LRP-2 can function to mediate cellular endocytosis of LDL, leading to its degradation. LRP-2 represents the second member of the LDLR family identified as functioning in the catabolism of LDL.

gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex.

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin.

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

Native TIMP-free 70 kDa progelatinase (MMP-2) secreted at elevated levels by RSV transformed fibroblasts.

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed.