Effects of acrylamide exposure on serum hormones, gene expression, cell proliferation, and histopathology in male reproductive tissues of Fischer 344 rats.

Acrylamide (AA) is a reactive monomer used in many technological applications, but it is the incidental formation during cooking of common starchy foods that leads to pervasive human exposure, typically in the range of 1 µg/kg body weight (bw)/day (d). AA is carcinogenic in multiple organs from both sexes of several rodent models and a consistent body of evidence points to a genotoxic mechanism based on metabolism to a DNA-reactive epoxide, glycidamide (GA). In F344 rats, tumorigenesis occurs in several hormonally regulated tissues (thyroid, mammary gland, and peri-testicular mesothelium), which has prompted speculation about endocrine dysregulation as a possible mechanism. The present study evaluated the effects of a 14 d exposure to AA administered through the drinking water on reproductive tissues and the hypothalamic-pituitary-testes (HPG) axis in male F344 rats. The doses selected encompass a range from approximately 2.5 mg/kg bw/d, which is carcinogenic after lifetime exposure, to 50 mg/kg bw/d, a maximally tolerable dose that causes hind limb paralysis. AA caused significant changes in serum hormones, histopathology, testicular gene expression, and cell proliferation, especially at the highest dose. Despite strong evidence for activation of the HPG axis subsequent to decreases in testosterone levels, and histopathological changes associated with significant effects on Leydig and germ cells, with concomitant mRNA expression changes, the precise mechanism(s) for AA-induced testicular toxicity remains unclear; however, the absence of evidence for increased proliferation of the peri-testicular mesothelium (Ki-67 immunoreactivity) does not support hormonal dysregulation as a contributing factor to the predisposition of this tissue to the carcinogenic effects of AA.

Cathepsin K inhibition reduces CTXII levels and joint pain in the guinea pig model of spontaneous osteoarthritis.

Cathepsin K is a cysteine proteinase which is believed to contribute to osteoarthritis (OA) pathogenesis. This brief report evaluates the effect of the novel selective cathepsin K inhibitor AZ12606133 on cartilage metabolism in the Dunkin-Hartley guinea pig model of spontaneous OA. In parallel, electrophysiological studies were performed to determine whether acute and chronic treatment with the cathepsin K inhibitor could alter joint nociception. Acute treatment of OA knees with AZ12606133 had no effect on joint afferent nerve activity; however, prolonged (1 month) administration of the cathepsin K inhibitor delivered via a chronically implanted osmotic pump significantly reduced mechanosensitivity in response to both non-noxious and noxious joint movements. Urinal concentrations of the cartilage breakdown products cross-linked C-telopeptides of type II collagen (CTXII) were also reduced by chronic cathepsin K inhibition. These data suggest that prolonged AZ12606133 administration can reduce cartilage turnover and joint nociception in the Dunkin-Hartley guinea pig model of spontaneous OA.

Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.

OBJECTIVE: To investigate the effects of a matrix metalloproteinase (MMP) inhibitor on joint pathology and pain behavior in the rat meniscal transection (MNX) model of osteoarthritis (OA) and evaluate which aspects of structural disease modification contribute to symptom improvement. METHODS: OA pathology was induced in male Lewis rats, by transecting the medial collateral ligament with (MNX) or without (SHAM) a full thickness cut through the meniscus. MNX animals were orally administered an equipotent MMP 2, 8, 9, 12, 13 inhibitor (0.25, 1 and 5 mg/kg/day) or vehicle from day 1. Chondropathy, osteophytosis, osteochondral vascularity were assessed from toluidine blue stained coronal sections of the total knee joint and weight-bearing asymmetry by incapacitance. Group differences were evaluated using 1-way analysis of variance (ANOVA) and associations as Spearman's correlation coefficients. RESULTS: Treatment with the MMP inhibitor reduced weight-bearing asymmetry from day 14 onwards, and attenuated chondropathy (both P<0.05). Osteochondral vascularity was elevated in MNX compared with SHAM-operated animals (P<0.001) and reduced, dose-dependently, by MMP inhibitor treatment (r=-0.89, P<0.05). Reduced osteochondral vascularity and chondropathy were associated with the amelioration of weight-bearing asymmetry (both P<0.05). CONCLUSION: Here we show that treatment with a MMP inhibitor reduces joint damage, osteochondral angiogenesis and behavioral evidence of pain. The association between osteochondral angiogenesis and pain behavior may be explained by perivascular nerve growth or stimulation of subchondral nerves following loss of osteochondral integrity. Our data suggest that targeting angiogenesis may have utility in the treatment of pain associated with structural damage in OA.

The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male Fischer 344 rats.

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Angiogenesis in two animal models of osteoarthritis.

OBJECTIVE: We have previously described angiogenesis at the osteochondral junction and in synovium of knees from patients with osteoarthritis (OA), but little is known about how closely animal models of OA resemble human disease with respect to vascular growth. This study aimed to characterise two animal models of knee OA with particular respect to osteochondral and synovial angiogenesis. METHOD: We examined the spontaneous Dunkin-Hartley (DH) guinea pig and medial meniscal transection (MNX) rat models of OA. Vessels at the osteochondral junction and in the synovium were identified by lectin immunohistochemistry and quantified by computer-assisted image analysis. Disease severity was assessed using a scoring system. RESULTS: Blood vessels crossed the osteochondral junction in juvenile rats and guinea pigs, with higher densities in the lateral than medial tibial plateau, the number decreasing with maturation in the absence of other OA changes. In the rat model, increased vascular density was observed both at the osteochondral junction and in the synovium, whilst osteochondral vascularity in control rats decreased with maturation, OA rats showed a persistence of blood vessels at the osteochondral junction. In rat synovium, blood vessel fractional area was increased in the hypertrophied synovium 14 days after surgery, then decreased to control levels by day 28. Significant differences in vascularity were not observed between affected (medial) and spared (lateral) compartments of guinea pig knees. CONCLUSION: The rat meniscal transection model of OA reproducibly displays both osteochondral and synovial angiogenesis comparable to our previous observations in human knee OA. DH guinea pigs, by contrast, display low vascularity throughout their protracted course of OA development. Changes in vascularisation occur early during the development of OA in the rat, and may contribute to the pathogenesis of OA.

A threshold neurotoxic amphetamine exposure inhibits parietal cortex expression of synaptic plasticity-related genes.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

The subjective experience of early postoperative pain following retrobulbar anaesthesia for enucleation and primary orbital implant.

INTRODUCTION: We performed a prospective audit of the level of postoperative pain experienced by patients following enucleation with insertion of a primary orbital implant after preincisional regional retrobulbar anaesthesia using bupivacaine 0.75% with 1:100,000 adrenaline. MATERIALS AND METHODS: An 11-point numerical ranking box scale was used to measure the subjective experience of postoperative pain following enucleation with insertion of a primary orbital implant in 40 patients with uveal melanoma. Surgery was performed under general anaesthesia with a supplementary peroperative retrobulbar injection of bupivacaine 0.75%/adrenaline 1:100,000. Pain scores were measured for the first 8 hours following administration of the block. RESULTS: The sample included 19 female and 21 male patients with a mean age of 66.7 years (31-87). At four hours post block, 80% were still pain free with 17% experiencing only mild to moderate pain (BS-11 = 1-4). Thirty-four (85%), twenty-eight (70%) and twenty-seven (67%) patients remained pain free at 2, 3 and 4 hours, respectively with no additional analgesia. The remainder scored BS-11 of 1-4 in 92% of cases. Twenty percent required supplementary analgesia (paracetemol in 78% cases) by 5 hours and 57% by 8 hours. BS-11 at 8 hours were 0 in 50%, 1-4 in 22% and 5-10 in 10% of patients (17% asleep). No complications using this technique were recorded. DISCUSSION: Using a preincisional retrobulbar injection of bupivacaine with adrenaline, BS-11 pain scores remained low with no or minimal additional analgesia for up to 4 hours post surgery. In combination with oral analgesia, effective pain control was provided in most cases for up to 8 hours post block.

Characterisation of the guinea pig model of osteoarthritis by in vivo three-dimensional magnetic resonance imaging.

OBJECTIVE: To characterise longitudinal changes in joint integrity and cartilage volume in vivo in the guinea pig spontaneous osteoarthritis (OA) model by magnetic resonance imaging (MRI). METHODS: Guinea pigs knee were imaged in vivo by high-resolution three-dimensional (3D) MRI between the ages of 3 and 12 months. Image analysis was performed to assess qualitative knee joint changes between 3 and 12 months (n=16) and quantitative volumetric changes of the medial tibial cartilage between 9 and 12 months (n=7). After imaging, animals were killed and knees were assessed macroscopically and histologically. RESULTS: From 3 to 6 months qualitative observation by MRI and histopathology indicated localised cartilage swelling on the medial tibial plateau. At 6 months, bone cysts had developed in the epiphysis. At 9 months, we observed by MRI and histopathology, fragmentation of the medial tibial cartilage in areas not protected by the meniscus. Cartilage degeneration had intensified at 12 months with evidence of widespread loss of cartilage throughout the tibial plateau. Segmentation of the MR cartilage images showed a 36% loss of volume between 9 and 12 months. CONCLUSIONS: We have achieved 3D image acquisition and segmentation of knee cartilage in a guinea pig model of chronic OA, which permits measurements previously only possible in man. High resolution and short acquisition time allowed qualitative longitudinal characterisation of the entire knee joint and enabled us to quantify for the first time longitudinal tibial cartilage volume loss associated with disease progression.

Visual complications of proton beam therapy for clival chordoma.

PURPOSE: Combining maximal surgical resection with high-dose proton radiation therapy is reported to be currently the best management of patients with clival chordoma. METHOD: Since 1991, four consecutive patients from our institution with this tumour have been referred for postoperative proton beam radiotherapy. RESULT: We have experienced an unusually high complication rate (50%) of delayed radiation optic neuropathy. This has resulted in profound, bilateral visual loss at 1 and 2 years postproton beam treatment. CONCLUSION: It has served as a reminder that proton beam therapy is not an innocuous treatment option and this devastating complication should be taken into account when discussing the management of clivus chordoma. We postulate whether the optic apparatus is particularly sensitive to proton vs photon damage.

Evaluation of methyl tert-butyl ether (MTBE) as an interference on commercial breath-alcohol analyzers.

Anecdotal reports suggest that high environmental or occupational exposures to the fuel oxygenate methyl tert-butyl ether (MTBE) may result in breath concentrations that are sufficiently elevated to cause a false positive on commercial breath-alcohol analyzers. We evaluated this possibility in vitro by establishing a response curve for simulated breath containing MTBE in ethanol. Two types of breath-alcohol analyzers were evaluated. One analyzer's principle of operation involves in situ wet chemistry (oxidation of ethanol in a potassium dichromate solution) and absorption of visible light. The second instrument uses a combination of infrared absorption and an electrochemical sensor. Both types of instruments are currently used, although the former method represents older technology while the latter method represents newer technology.The percent blood alcohol response curve was evaluated over a breath concentration range thought to be relevant to high-level environmental or occupational exposure (0-361 microg/l). Results indicate that MTBE positively biases the response of the older technology Breathalyzer when evaluated as a single constituent or in combination with ethanol. We conclude that a false positive is possible on this instrument if the MTBE exposure is very high, recent with respect to testing, and occurs in combination with ethanol consumption. The interference can be identified on the older technology instrument by a time dependent post-reading increase in the instrument response that does not occur for ethanol alone. In contrast, the newer technology instrument using infrared and electrochemical detectors did not respond to MTBE at lower levels (0-36 microg/l), and at higher levels (>72 microg/l) the instrument indicated an "interference" or "error". For this instrument, a false positive does not occur even at high MTBE levels in the presence of ethanol.

L-ephedrine-induced neurodegeneration in the parietal cortex and thalamus of the rat is dependent on hyperthermia and can be altered by the process of in vivo brain microdialysis.

Multiple doses of the dietary supplement L-ephedrine can cause severe hyperthermia and modest dopamine depletions in the rat brain. Since D-amphetamine treatment can result in neurodegeneration, the potential of L-ephedrine to produce similar types of degeneration was investigated. Adult male rats, some implanted in the caudate/putamen (CPu) for microdialysis, were given four doses of 25 mg/kg L-ephedrine or 5 mg/kg D-amphetamine (2 h between doses) at an ambient temperature of 23 degrees C. L-ephedrine-induced degeneration in the forebrain was dependent on the degree of hyperthermia. Layer IV of the parietal cortex was the most sensitive to L-ephedrine treatment with peak body temperatures of at most 40.0 degrees C necessary to produce degeneration. Extensive neurodegeneration in the parietal cortex after L-ephedrine treatment was as pronounced as that previously described for D-amphetamine treatment and also occurred in the intralaminar, ventromedial and ventrolateral thalamic nuclei in rats with severe hyperthermia (peak body temperatures>41.0 degrees C). The neurodegeneration induced by L-ephedrine may have resulted in part from excitotoxic mechanisms involving the indirect pathways of the basal ganglia and related areas. No differences were observed between microdialysis and non-implanted rats with respect to degree of tyrosine hydroxylase (TH) loss in the CPu after either D-amphetamine or L-ephedrine treatment. However, neurodegeneration resulting from D-amphetamine and L-ephedrine was reduced in the microdialysis animals in the hemisphere ipsilateral to the probe, which raises concerns when using the technique of in vivo microdialysis to evaluate neurodegeneration. The results of this study, in conjunction with human clinical evaluation of ephedrine neurotoxicity, indicate that regionally specific damage may occur in the cortex of some humans exposed to ephedrine in the absence of stroke or hemorrhage.

Phenobarbital and dizocilpine can block methamphetamine-induced neurotoxicity in mice by mechanisms that are independent of thermoregulation.

Body temperature profiles observed during methamphetamine (METH) exposure are known to affect dopamine and tyrosine hydroxylase (TH) levels in the striatum of mice; hyperthermia potentiates depletion while hypothermia is protective against depletions. In the current study, the doses of METH were sufficiently great that significant dopamine and TH depletions occurred even though hypothermia occurred. Four doses, administered at 2 h intervals, of 15 mg/kg (4x15 mg/kg) D-METH significantly decreased TH and dopamine levels to 50% of control in mice becoming hypothermic during dosing in a 13 degrees C environment. Phenobarbital or dizocilpine during METH exposure blocked the depletions while diazepam did not. Phenobarbital and dizocilpine did not block depletions by altering the hypothermic profiles from that observed during METH only exposure. Here we show that phenobarbital and dizocilpine can block measures of METH neurotoxicity by non-thermoregulatory mechanisms.

Seizure activity and hyperthermia potentiate the increases in dopamine and serotonin extracellular levels in the amygdala during exposure to d-amphetamine.

Behavioral stereotypy, hyperthermia, and convulsive activity produced by exposure to multiple doses of d-amphetamine (AMPH) were related to changes in the extracellular levels of dopamine and serotonin (5-HT) in the amygdala, using the technique of microdialysis in awake and freely moving rats. Hyperactivity and stereotypy, as well as increases in microdialysis dopamine levels ranging from 100-300% of pre-AMPH basal microdialysate levels (BL), occurred during exposure to 3 doses of 2.5 mg/kg (3 x 2.5 mg/kg) AMPH. Three doses of 5 mg/kg produced a more intense stereotypic behavior as well as hyperthermia, and resulted in large increases in the peak dopamine levels (700% BL) while 5-HT levels were increased to a lesser extent (300% BL). The highest doses tested of 3 x 15 mg/kg produced convulsive activity, seizures, intense stereotypy and hyperthermia with peak microdialysate dopamine (1300% BL) and 5-HT levels (1800% BL) that were 2-fold and 6-fold greater, respectively, than those at the 3 x 5-mg/kg doses. Microdialysate glutamate levels were not changed by AMPH exposure. Rats that did not become hyperthermic when dosed with 15 mg/kg AMPH in a cold environment (10 degrees C) exhibited some hyperactivity and stereotypic behavior, but not overt convulsive behavior. Dopamine and 5-HT levels in these rats were significantly reduced by about 75% and 60%, respectively, compared to the room-temperature group. Inclusion of 2 microM tetrodotoxin (TTX) in the microdialysis buffer significantly reduced the 15-mg/kg AMPH-induced increases in dopamine by 30% and the increase in 5-HT levels by 70% at room temperature. These results indicate that a smaller portion of the dopamine release evoked by doses of AMPH that induce seizure activity is neuronal impulse-dependent while the majority of 5-HT released is impulse-dependent. Irrespective of impulse activity, the hyperthermia alone markedly potentiated dopamine release but had a lesser effect on 5-HT release. Thus, there are differences in the regulation of dopamine and serotonin release in the amygdala from high doses of AMPH, which are known to produce neurotoxicity. Further studies are necessary to determine the impact of these differences in release on AMPH neurotoxicity.

The use of the fluorescein disappearance test in the management of childhood epiphora.

PURPOSE. The fluorescein disappearance test (FDT) is reported to be an objective measure of lacrimal outflow in congenital non-canalisation of the nasolacrimal system. We introduced FDT into our evaluation of children with epiphora to investigate its sensitivity/specificity with regard to symptoms in a prospective study. We also examined the FDT against findings at syringing and probing (S&P) and persistence or resolution of symptoms on follow-up. METHODS. Over a period of 16 months, 88 consecutive children (median age 12 months; range 2.5-192) with epiphora were reviewed and FDT measured at 5 and 10 minutes; 66 FDT were evaluated for inter-observer variation in a masked fashion. S&P were scheduled on the basis of symptoms, abnormal FDT and age "1 year. Normal FDT prompted review. Younger children were observed for natural history and possible resolution with repeat FDT. Equivocal symptoms and normal FDT initiated discharge and telephone review. RESULTS. Inter-observer correlation coefficient = 0.86. Sensitivity/specificity of FDT was 76/76% at 5 minutes and 63/89% at 10 minutes, respectively. In patients under 1 year of age undergoing follow-up (29 patients; 38 eyes) FDT at 10 minutes correctly predicted persistence of symptoms in 65% and resolution in 66% of eyes (follow-up 3-14 months; mean 6); 23 patients (mean age 27 months; range 12-72) underwent S&P with positive surgical findings in 20 (87%). On review, symptoms had improved in 64% eyes (20/31). CONCLUSION. FDT at 5 minutes is an objective measure of symptoms with high inter-observer agreement and agreement with surgical findings. FDT read at 10 minutes may be useful to indicate the persistence or resolution of symptoms and guide patient management.

Neuronal degeneration in the limbic system of weanling rats exposed to saline, hyperthermia or d-amphetamine.

Neuronal degeneration was detected in the tenia tecta and other regions of the anterior limbic system of male weanling rats 3 days after four doses of 5 mg/kg d-amphetamine (4 x 5 mg/kg AMPH) when seizures occurred during AMPH exposure. Neurodegeneration in the parietal cortex, loss of tyrosine hydroxylase immunoreactivity in the caudate-putamen (CPu) and decreases in CPu tissue dopamine levels in weanlings was much less than those previously observed in adults. The neurotoxicity seen in the parietal cortex and CPu of the weanlings was much less than previously seen in adults even though severe hyperthermia and the behavior of retrograde propulsion occurred during AMPH exposure. Neurodegeneration was not detected in any of the previously mentioned brain regions in controls and weanlings made hyperthermic by a warm environment. However, signs of spontaneous neurodegeneration were seen in the posterior piriform cortex (Pir), posteriolateral cortical amygdaloid nucleus (PLCo), and the amygdalopiriform transition area (APir) of control weanlings. The doses of AMPH and the degree of hyperthermia necessary to induce seizures were substantially lower in weanlings compared to those previously observed in adult rats. Further studies will be necessary to determine if the susceptibility of weanlings to AMPH-induced seizures is related to or dependent on the same processes involved in producing degeneration in the posterior limbic system of saline controls.

Diverse, yet-to-be-cultured members of the Rubrobacter subdivision of the Actinobacteria are widespread in Australian arid soils.

Phylogenetic analyses of ribosomal RNA gene sequences (rDNAs) retrieved from an Australian desert soil sample (Sturt National Park) revealed the presence of a number of clones which branched deeply from the high GC Gram-positive division line of descent. The most abundant group of these clones were related to Rubrobacter. An oligonucleotide probe was designed to have broad specificity to Rubrobacter and relatives. This probe was used to interrogate eight rDNA libraries representing four distinct land forms within the Australian arid zone. Relative abundance of Rubrobacter-relatives in these samples ranged from 2.6 to 10.2%. Clones from these libraries were selected for sequence analysis on the basis of a heteroduplex mobility assay to maximise the diversity represented in the sample. Phylogenetic analyses of these rDNA clones and Rubrobacter-related clones reported in the literature show strong support for three distinct groups. Database-searching revealed 'Rubrobacteria' were relatively abundant in a number of published soil rDNA libraries but absent from others. A PCR assay for group-1 'Rubrobacteria' was used to test for their presence in 21 environmental samples. Only marine and arid-zone soil samples gave positive PCR results. Taken together these results indicate 'Rubrobacteria' are a widespread group of variable abundance and diversity.

Proteolysis of SpolVB is a critical determinant in signalling of Pro-sigmaK processing in Bacillus subtilis.

SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K). We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.