BRAF-mediated brain tumors in adults and children: A review and the Australian and New Zealand experience.

The mitogen-activated protein kinase (MAPK) pathway signaling pathway is one of the most commonly mutated pathways in human cancers. In particular, BRAF alterations result in constitutive activation of the rapidly accelerating fibrosarcoma-extracellular signal-regulated kinase-MAPK significant pathway, leading to cellular proliferation, survival, and dedifferentiation. The role of BRAF mutations in oncogenesis and tumorigenesis has spurred the development of targeted agents, which have been successful in treating many adult cancers. Despite advances in other cancer types, the morbidity and survival outcomes of patients with glioma have remained relatively stagnant. Recently, there has been recognition that MAPK dysregulation is almost universally present in paediatric and adult gliomas. These findings, accompanying broad molecular characterization of gliomas, has aided prognostication and offered opportunities for clinical trials testing targeted agents. The use of targeted therapies in this disease represents a paradigm shift, although the biochemical complexities has resulted in unexpected challenges in the development of effective BRAF inhibitors. Despite these challenges, there are promising data to support the use of BRAF inhibitors alone and in combination with MEK inhibitors for patients with both low-grade and high-grade glioma across age groups. Safety and efficacy data demonstrate that many of the toxicities of these targeted agents are tolerable while offering objective responses. Newer clinical trials will examine the use of these therapies in the upfront setting. Appropriate duration of therapy and durability of response remains unclear in the glioma patient cohort. Longitudinal efficacy and toxicity data are needed. Furthermore, access to these medications remains challenging outside of clinical trials in Australia and New Zealand. Compassionate access is limited, and advocacy for mechanism of action-based drug approval is ongoing.

First use of artificial canopy bridge by the world's most critically endangered primate the Hainan gibbon Nomascus hainanus.

All gibbon species (Primates: Hylobatidae) are facing high extinction risk due to habitat loss and hunting. The Hainan gibbon Nomascus hainanus is the world's most critically endangered primate, and one of the priority conservation actions identified is to establish artificial canopy corridors to reconnect fragmented forests. The effectiveness of artificial canopy bridge as a conservation tool for wild gibbons has not been widely tested, and the results are rarely published. We constructed the first canopy bridge for Hainan gibbon in 2015 to facilitate passage at a natural landslide; mountaineering-grade ropes were tied to sturdy trees with the help of professional tree climbers and a camera trap was installed to monitor wildlife usage. Hainan gibbon started using the rope bridge after 176 days, and usage frequency increased with time. All members in the gibbon group crossed the 15.8 m rope bridge except adult male. Climbing was the predominant locomotor mode followed by brachiation. This study highlights the use and value of rope bridges to connect forest gaps for wild gibbons living in fragmented forests. While restoring natural forest corridors should be a priority conservation intervention, artificial canopy bridges may be a useful short-term solution.

A new phytotelm-breeding treefrog of the genus Nasutixalus (Rhacophoridae) from western Yunnan of China.

A new treefrog of the genus Nasutixalus is described from montane evergreen forest in Yingjiang County, in western Yunnan, China. The new species, Nasutixalus yingjiangensis sp. nov., can be distinguished from its congeners by a combination of the following characters: medium body size (SVL 39.5-40.0 mm in adult males, 47.5 mm in a single female); tympanum indistinct and covered with tubercles; disc diameter of third finger greater than tympanum diameter; dorsal skin relatively smooth, scattered with small tubercles, those on head and anterior dorsum of body more dense and more prominent; light brown above with a dark brown marking between eyes and two broad dark brown lateral strips on the dorsum; iris with a weak "X"-shaped, light colored marking; interorbital distance shorter than the upper eyelid width; comparatively short foot (mean TFL/SVL ratio 67.0% and 62.9% in males and female respectively). The new species appears to be forest-dependent and was found in well-preserved montane rainforest; it inhabits the canopy and breeds in tree holes during the rainy season. The type locality of the new species is an isolated forest fragment surrounded by degraded landscape and efforts are already underway to protect the site. Nasutixalus yingjiangensis sp. nov. represents the third known species of the newly established genus Nasutixalus.

Discontinuities and disruptions in drug dosage guidelines for the paediatric population.

AIMS: This study investigates paediatric drug dosage guidelines with the aim of investigating their agreement with body surface area (BSA) scaling principles. METHODS: A total of 454 drug dosage guidelines listed in the AMH-CDC 2015 were examined. Data extracted included the administration, frequency and dose per age bracket from 0 to 18 years. Drug treatments were categorized as follows: (1) The same dose recommendation in milligrams per kilogram (mg kg-1 ) for all age/weights; (2) Change in the mg kg-1 dosing according to age/weight; (3) Change in dose in mg according to age/weight; (4) Change from mg kg-1 dosing to a dose in mg according to age/weight; (5) The same recommendation for all age/weight groups in mg; or (6) BSA dosing. Example drugs were selected to illustrate dose progression across ages. RESULTS: Most drug treatments (63%) have the same mg kg-1 dose for all age/weight groups, 14% are dosed in mg kg-1 across all ages with dose changes according to age/weight, 13% were dosed in mg across all ages with dose changes, 10% switched from mg kg-1 to a set dose in mg, 4.2% have the same dose in mg for all age and weight groups and 2.2% are dosed according to BSA. CONCLUSIONS: Paediatric dosage guidelines are based on weight-based formulas, available dosing formulations and prior patterns of use. Substantial variation from doses predicted by BSA scaling are common, as are large shifts in recommended doses at age thresholds. Further research is required to determine if better outcomes could be achieved by adopting biologically based scaling of paediatric doses.

Rediscovery of the sun bear (Helarctos malayanus) in Yingjiang County,Yunnan Province,China.

The sun bear, Helarctos malayanus (Raffles, 1821), is a forest-dependent bear species distributed in tropical Southeast Asia. The species was previously reported from scattered localities in southwestern China, which is at the northeastern edge of its global range. Due to the scarcity of reliable recent records, some authorities cast doubt on the continued existence of sun bear in China. Here we present the rediscovery of this species in Yingjiang County, western Yunnan Province, China, near the international border with Myanmar's Kachin State.

Medicine information exchange networks among healthcare professionals and prescribing in geriatric medicine wards.

AIMS: Effective transfer of information is vital for rational drug therapy. This is particularly important for older patients, who have a high prevalence of polypharmacy and are managed by multidisciplinary teams. We aimed to assess medicine information exchange (MIE) networks in geriatric medicine wards and whether they are associated with prescribing patterns. METHODS: We conducted network analysis in acute geriatric medicine wards from four hospitals to characterize MIE networks among multidisciplinary team members. Corresponding patient data were collected to analyze high-risk prescribing in conjunction with network characteristics. RESULTS: We found that junior doctors, senior nurses and pharmacists were central to MIE across all four hospitals. Doctors were more likely than other professions to receive medicines information in three hospitals. Reciprocity and the tendency to communicate within one's own profession also influenced network formation. No difference was observed in prescribing practice between hospitals. CONCLUSIONS: Understanding MIE networks can identify gaps in multidisciplinary communication that can be addressed. Networks may identify targets for dissemination of interventions to improve prescribing.

How many remnant gibbon populations are left on Hainan? Testing the use of local ecological knowledge to detect cryptic threatened primates.

For Critically Endangered "species of extreme rarity," there is an urgent need to clarify the potential survival of remnant populations. Such populations can be difficult to detect using standard field methods. Local ecological knowledge (LEK) represents an important alternative source of information, but anecdotal reports of rare or possibly extinct species can contain uncertainty and error. The Hainan gibbon (Nomascus hainanus), the world's rarest primate species, is confirmed to only survive as a tiny remnant population in Bawangling National Nature Reserve, China, but unverified gibbon sightings have been reported from other forest areas on Hainan. We conducted a large-scale community interview survey to gather new data on patterns of primate LEK from 709 respondents around seven reserves across Hainan, to investigate the possibility of gibbon survival outside Bawangling and assess whether LEK can provide useful information for conservation management of cryptic remnant populations. Comparative LEK data for gibbons and macaques are consistent with independent data on the relative status of these species across Hainan. Local awareness and experience of gibbons was low across Hainan, including at Bawangling, but we recorded recent anecdotal gibbon reports from most reserves. A follow-up field survey at Limushan Provincial Nature Reserve did not detect gibbons, however, and documented intensive wildlife exploitation within this reserve. All other surveyed landscapes showed some statistically lower levels of respondent awareness, experience, or sighting histories of gibbons compared to Bawangling, and are therefore considered biologically unlikely to support gibbons. Unverified LEK data can provide important insights into the possible status of cryptic remnant populations when assessed carefully and critically in relation to data from known populations.

A new species of the genus Leptobrachium (Anura: Megophryidae) from the Gaoligongshan Mountain Range, China.

We describe a new species of the genus Leptobrachium from the Gaoligongshan Mountain Range, Yunnan Province of China based on molecular and morphological evidences. The new species, Leptobrachium tengchongense sp. nov., can be distinguished from its congeners by a combination of the following characters: (1) relatively small size (adult males SVL 41.7-51.5 mm); (2) head width slightly larger than head length; (3) tympanum indistinct; (4) two palmar tubercles oval and distinct, inner one larger than outer one; (5) sexually active males without spines on the upper lip; (6) dorsal skin smooth with distinct network of ridges; (7) dorsum pinkish grey and scattered with irregular black markings; (8) venter dark purplish-gray with numerous small white spots on tubercles, solid white chest; (9) iris bicolored, upper one-third light blue, lower two-third dark brown. With the description of the new species, the number of Leptobrachium species currently known from China adds up to ten.

Two new species of the genus Goniurosaurus (Squamata: Sauria: Eublepharidae) from southern China.

Two new species of large geckos in the genus Goniurosaurus are described based on specimens collected from karst areas of Guangxi Zhuang Autonomous Region, southern China: Goniurosaurus kadoorieorum sp. nov. and Goniurosaurus kwangsiensis sp. nov. Data on natural history of the new species are provided, as well as discussions on the current conservation status of Goniurosaurus species in southern China. Due to the popularity of this genus as novelty pets, and recurring cases of scientific descriptions driving herpetofauna to near-extinction by commercial collectors, we do not disclose the collecting localities of these restricted-range species in this publication. However, such information has been presented to relevant government agencies, and is available upon request by fellow scientists.

NMR-based urinalysis for rapid diagnosis of ß-ureidopropionase deficiency in a patient with Dravet syndrome.

BACKGROUND: Beta-ureidopropionase deficiency is a rare inborn error of metabolism (IEM) affecting pyrimidine metabolism. To-date, about 30 genetically confirmed cases had been reported. The clinical phenotypes of this condition are variable; some patients were asymptomatic while some may present with developmental delay or autistic features. In severe cases, patients may present with profound neurological deficit including hypotonia, seizures and mental retardation. Using NMR-based urinalysis, this condition can be rapidly diagnosed within 15 min. CASE: An 11-month-old Chinese boy had dual molecular diagnoses, ß-ureidopropionase deficiency and Dravet syndrome. He presented with intractable and recurrent convulsions, global developmental delay and microcephaly. Urine organic acid analysis using GC-MS and NMR-based urinalysis showed excessive amount of ß-ureidopropionic acid and ß-ureidoisobutyric acid, the two disease-specific markers for ß-ureidopropionase deficiency. Genetic analysis confirmed homozygous known disease-causing mutation UPB1 NM_016327.2: c.977G>A; NP_057411.1:p.R326Q. In addition, genetic analysis for Dravet syndrome showed the presence of heterozygous disease-causing mutation SCN1A NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2. CONCLUSIONS: The differentiation between Dravet syndrome and ß-ureidopropionase deficiency is clinically challenging since both conditions share overlapping clinical features. The detection of urine ß-ureidoisobutyric and ß-ureidopropionic acids using NMR or GC-MS is helpful in laboratory diagnosis of ß-ureidopropionase deficiency. The disease-causing mutation, c.977G>A of ß-ureidopropionase deficiency, is highly prevalent in Chinese population (allele frequency=1.7%); ß-ureidopropionase deficiency screening test should be performed for any patients with unexplained neurological deficit, developmental delay or autism.

A new species of the genus Opisthotropis Gunther, 1872 (Squamata: Colubridae: Natricinae) from Guangdong Province, China.

A new natricid snake of the genus Opisthotropis Gunther, 1872, Opisthotropis laui sp. nov., is described from Mt. Gudou, Jiangmen City, Guangdong Province, China. The new species can be distinguished from other congeners by the combination of the following characters: dorsal scales weakly keeled throughout, in 25:23:23 rows; 10 supralabials; 11 infralabials; two internasals, longer than wide, not touching the loreal; one loreal, not touching the eye; one preocular; two postoculars; one anterior temporal scale; 152 ventrals; 53 subcaudals; body and tail dark olive above, with light yellow crossbars.

Mycobacterium tuberculosis H37Rv sigC is expressed from two promoters but is not auto-regulatory.

The extracytoplasmic function (ECF) sigma factor SigC has been implicated in the pathogenesis of Mycobacterium tuberculosis but control of its expression and activity is poorly understood. No proteins that interact with SigC have been detected leading to the suggestion that this sigma factor may be primarily controlled at the level of transcription. It has been suggested that SigC may be autoregulatory and a role has also been proposed for SigF in the expression of sigC. In this study we identified two promoters that were active under standard growth conditions by a combination of transcript start site mapping and promoter-lacZ fusion assays. The dominant promoter, P1, closely resembled mycobacterial SigA-dependent promoters, and introduction of a single base change at the conserved A of the -10 region eliminated promoter activity. Although the sequence of the other, P2, closely resembled the reported SigC consensus motifs, expression directed by this promoter was unaltered in a ΔsigC mutant strain, or in strains defective in other ECF sigma factors for which some similarity in consensus sequences was apparent. Comparison of the effects of different changes in the -10 region suggested that the P2 promoter was most likely recognised by SigA.

Evaluation of sleep disturbances in children with epilepsy: a questionnaire-based case-control study.

Epilepsy is a chronic neurological disorder accompanied by a wide range of comorbid conditions that can adversely affect the quality of life of children. Sleep disturbances not only predispose children to mood, cognitive, and behavioral impairments, but also have a significant impact on physical health. The aim of this study was to evaluate sleep patterns among Chinese children with epilepsy and healthy subjects in Hong Kong, and examine the relationship between parent-reported sleep problems and specific epilepsy parameters. We conducted a cross-sectional, questionnaire-based, case-control study and included 63 children with epilepsy and 169 healthy children aged between 4 and 12 years. The Children's Sleep Habits Questionnaire (CSHQ) was used as an assessment tool. Our results indicated that children with epilepsy have similar sleep patterns but greater sleep disturbances compared with healthy subjects. Sleep problems should not be overlooked, and a comprehensive review of the sleep habits of this group of patients should be conducted.

Mycobacterium smegmatis expressing a chimeric protein MPT64-proteolipid protein (PLP) 139-151 reorganizes the PLP-specific T cell repertoire favoring a CD8-mediated response and induces a relapsing experimental autoimmune encephalomyelitis.

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139-151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS(p139)). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS(p139) was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS(p139) modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4(+) T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS(p139) because lymph node APCs infected with rMS(p139) selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS(p139) expanded p139-specific CD8(+) cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross-reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

The extracellular domain of CD11d regulates its cell surface expression.

A mAb targeting the CD11d subunit of the leukocyte integrin CD11d/CD18 decreases intraspinal inflammation and oxidative damage leading to improved neurological outcomes in rodent models of SCI. CD11d/CD18 is the fourth member of the beta2-integrin family. Current evidence indicates that CD11d/CD18 is regulated differently than other beta2-integrins, suggesting that CD11d(+) leukocytes play a distinct role in inflammation. Although the transcriptional control of CD11d expression has been evaluated, control of the intracellular distribution of CD11d has not been addressed. For this reason and as a result of the potential of CD11d as a therapeutic target for SCI and possibly other CNS injuries, we investigated the intracellular localization and surface expression of CD11d in cultured cells. CD11d and CD18 were fused at their C-termini with YFP and mRFP, respectively. Flow cytometry and confocal microscopy demonstrated that rCD11d-YFP is expressed on the cell surface of leukocyte cell lines expressing CD18. In contrast, in heterologous cell lines, CD11d-YFP is retained intracellularly in the TGN. Coexpression of CD11d-YFP and CD18-mRFP relieves this intracellular restriction and allows the CD11d/CD18 heterodimer to be surface-expressed. Based on domain-swapping experiments with CD25, the extracellular domain of CD11d is required and sufficient for the observed intracellular retention in heterologous cells. Furthermore, the transmembrane and C-terminus are also required for proper heterodimerization with CD18 and localization to the plasma membrane. These findings suggest that multiple CD11d domains play a role in controlling intracellular location and association with CD18.

Src and FAK mediate cell-matrix adhesion-dependent activation of Met during transformation of breast epithelial cells.

Cell-matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with beta1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell-matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells.

Coordinated integrin and growth factor regulation of primary keratinocyte migration mediated through extracellular signal regulated kinase and phosphoinositide 3-kinase.

We have examined coordinated integrin and growth factor regulation of primary keratinocyte migration mediated by phosphoinositide 3-kinase (PI3K) and mitogen-activated extracellular-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK). On collagen I and fibronectin substrates, both epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulated chemokinetic (random) and chemotactic (directional) migration. On provisional matrix, a combination of fibronectin and fibrin found in the early phase of wound healing, EGF and HGF-stimulated significant chemotactic but little or no chemokinetic cell movement. Blocking mAbs to integrin alpha2beta1 and alpha5beta1 effectively inhibited EGF- and HGF-stimulated chemokinetic and chemotactic cell movement on collagen I and fibronectin, respectively; however, HGF-stimulated chemotactic migration on collagen I was only partially inhibited by alpha2beta1 blocking mAb. Differentiated keratinocytes underwent reduced chemokinetic and chemotactic migration compared with undifferentiated keratinocytes; however, EGF-stimulated migration was reduced more than HGF-stimulated migration. When the migratory response on collagen I and fibronectin was assessed in the presence of the MEK-specific inhibitor PD98059, EGF- and HGF-stimulated chemotaxis was significantly reduced, whereas PD98059 had little effect on the stimulated chemokinesis. PI3K-specific inhibitor LY294002 reduced EGF- and HGF-stimulated chemokinesis and chemotaxis on collagen I and fibronectin. Thus beta1 integrins acted in concert with EGF and HGF to regulate migration of primary keratinocytes on extracellular matrix components via PI3K and MEK/ERK.

Lipid incorporation inhibits Src-dependent assembly of fibronectin and type I collagen by vascular smooth muscle cells.

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and alpha5beta1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

Cathepsin B is involved in the trafficking of TNF-alpha-containing vesicles to the plasma membrane in macrophages.

TNF-alpha is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-alpha, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-alpha production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-alpha in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-alpha than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-alpha-containing vesicle trafficking to the plasma membrane.

Interaction of epidermal growth factor, Ca2+, and matrix metalloproteinase-9 in primary keratinocyte migration.

Elevations of epidermal growth factor (EGF) and Ca(2+) concentrations in the wound site are associated with reepithelialization during wound healing. In addition, Ca(2+) and EGF can both induce increases in matrix metalloproteinase-9 (MMP-9) synthesis. However, little is known about the interplay of these events in regulating the migration properties of primary keratinocytes on collagen I, the most abundant extracellular matrix component in the skin. We found that EGF stimulated both chemokinetic and chemotactic migration of primary keratinocytes on collagen I; however, MMP-9 was required for EGF-stimulated chemotaxis but not EGF-stimulated chemokinesis. Calcium at 0.5 mM stimulated chemokinetic migration of keratinocytes. Together, Ca(2+) and EGF stimulated higher levels of chemokinesis than either stimulus alone. Furthermore, Ca(2+) could restore the ability of keratinocytes from MMP-9 null mice to undergo EGF-stimulated chemotaxis. The phosphatidylinositol-3 kinase inhibitor LY294002 inhibited both EGF- and Ca(2+)-stimulated chemokinetic migration. In contrast, the MEK inhibitor PD98059 blocked Ca(2+)- but not EGF-stimulated chemokinetic migration of keratinocytes. A combination of PD98059 and LY294002 was required to inhibit Ca(2+) enhancement of EGF-stimulated migration completely. Calcium-stimulated chemokinesis was completely blocked by either the protein kinase C-alpha inhibitor Gö6976 or the src/fyn inhibitor PP2. Using primary keratinocytes, our results showed how the combined action of Ca(2+), EGF, and MMP-9 regulated the contributions of extracellular-regulated kinase and phosphatidylinositol-3 kinase toward chemokinetic and chemotactic migration of keratinocytes.