Antimicrobial resistance tendency and collateral sensitivity of Staphylococcus aureus adapted to antibiotics or extracts of medicinal plants grown in Viet Nam.

Antimicrobial resistance has silently turned into one of the biggest threats to global health, marking the fall of the Golden age of antibiotics. In the search for antibiotic replacement or enhancement, plant-derived natural compounds have attracted lots of interest. Even though firmly believed, the low-resistance tendency of pathogenic bacteria against plant extracts has been scarcely demonstrated. In this study, we investigated the antibacterial activities of diethyl ether extracts from six medicinal plants grown in Viet Nam against Staphylococcus aureus and its variants, which were in vitro adapted to the same extracts. After 30 passages of S. aureus growing under sub-lethal concentrations of plant extracts or antibiotics, the bacteria slowly adapted to the extracts while rapidly resisting the antibiotics. Most of the resulting strains obtained from the adaptation to plant extracts were collaterally sensitive to antibiotics. In contrast, antibiotic-adapted strains showed cross-resistance to both antibiotics and extracts. The findings provided evidence of the low-resistance tendency of S. aureus to antimicrobial plant extracts. It is the first time a collateral antibiotic sensitivity of S. aureus adapted to natural compounds has been observed, suggesting an alternative approach to fight antibiotic resistance.

Effects of sea-level rise on physiological ecology of populations of a ground-dwelling ant.

INTRODUCTION: Sea-level rise is a consequence of climate change that can impact the ecological and physiological changes of coastal, ground-dwelling species. Sea-level rise has a potential to inundate birds, rodents, spiders, and insects that live on the ground in coastal areas. Yet, there is still much to be learned concerning the specifics of these impacts. The red imported fire ant Solenopsis invicta (Buren) excavates soil for its home and is capable of surviving flooding. Because of their ground-dwelling life history and rapid reproduction, fire ants make an ideal model for discovery and prediction of changes that may be due to sea-level rise. There are up to 500,000 individuals in a colony, and these invasive ants naturally have a painful sting. However, observations suggest that colonies of fire ants that dwell in tidally-influenced areas are more aggressive with more frequent stings and more venom injected per sting (behavioral and physiological changes) than those located inland. This may be an adaption to sea-level rise. Therefore, the objective of this study is to elucidate differences in inland and coastal defensiveness via micro-dissection and comparison of head width, head length, stinger length, and venom sac volume. But first because fire ants' ability to raft on brackish tidal water is unknown, it had to be determined if fire ants could indeed raft in brackish water and examine the behavior differences between those flooded with freshwater vs. saltwater. METHODS: To test the coastal-aggression hypothesis, inland colonies and coastal colonies, which experience relatively greater amounts of flooding, specifically regular tidal and windblown water and oscillations (i.e. El Nino Southern Oscillation) from the Gulf of Mexico, were collected. To mimic sea-level rise, the colonies were flooded in salinities that correspond to both their collection site and conditions found in a variety of locales and situations (such as storm surge from a tropical storm). Individual ants were immediately taken from each colony for dissection before flooding, 1-hour into flooding, and 24-hours into flooding. RESULTS AND DISCUSSION: Fire ants use their venom to defend themselves and to communicate alarm or aggression. Dissections and measurement of heads, venom sacs, and stingers revealed both coastal and inland colonies experience an increase in venom sac volume after 24 hours; in fact coastal colonies increased their venom volume by 75% after 24 h of flooding Whether this venom sac enlargement is due to diffusion of water or venom sac production is unknown. These ground-dwelling ants exhibit physiological and behavioral adaptations to ongoing sea-level rise possibly indicating that they are responding to increased flooding. Fire ants will raft on high-salinity water; and sea-level rise may cause stings by flooded ants to be more severe because of increased venom volume.

Laparoscopic living donor right nephrectomy: Assessment of outcome and association of BMI to length of right renal vein. / Nefrectomía laparoscópica derecha de donante vivo: evaluación del resultado y asociación del IMC con la longitud de la vena renal derecha.

AIMS: The aim of this study was to describe outcomes of laparoscopic living donor right nephrectomy (LLDRN) and study factors affecting the length of right renal vein from the donors. MATERIAL AND METHODS: This study was conducted in 60 donors (48 males and 12 females) from January 2016 to December 2017. We performed a retrospective review of consecutive patients who underwent transperitoneal right laparoscopic living donor nephrectomy at our unit. RESULTS: LLDRN was successfully performed in all subjects by the same surgeons. Among 60 cases, 47 donors had single renal artery and vein, 2 cases had one artery and 2 veins, and 5 donors had 2 arteries and one vein, and the rest had 2-3 arteries with 1-3 veins. Operative time was 142.60±33.73min. Warm ischemic time was 2.64±0.76min. The mean hospital stay was 6.69±0.63 days. The median length of right renal vein was 1.92±0.41cm. All transplanted kidneys showed immediate function. No graft losses were recorded. Almost no gender differences were found in study variables except BMI and warm ischemic time, that was higher BMI but shorter warm ischemic time in female versus male donors. Further analysis showed a negative correlation between BMI and right renal vein (r=-0.282, P<0.05), but a positive correlation between operative time and estimate blood loss (r=0.37, P<0.01). CONCLUSIONS: LLDRN is a feasible safe procedure, less traumatic approach, and provides good outcomes kidney for recipients. Notably, in the study group the higher BMI was associated with resulting more difficult LLDRN and kidney transplantation.

Alterations in the Serotonin and Dopamine Pathways by Cystathionine Beta Synthase Overexpression in Murine Brain.

Cystathionine beta synthase (CBS) is one of the 225 genes on chromosome 21 (HSA 21) that are triplicated in persons with trisomy 21 (Down syndrome). Although most triplicate HSA21 genes have their orthologous genes on murine chromosome 16, the murine ortholog of hCBS is on murine chromosome 17 and thus is not present in the well-studied Ts65Dn mouse model of trisomy 21. Persons with trisomy 21 (T21) present deficits in neurotransmission and exhibit early brain aging that can partially be explained by monoamine neurotransmitter alterations. We used transgenic mice for the hCBS gene, which overexpress the CBS protein in various brain regions, to study if CBS overexpression induces modifications in the monoamine neurotransmitters in the hypothalamus, thalamus, hippocampus, and striatum from transgenic and control female and male mice aged 3-4 months and 11-12 months. Sex, age, and brain area each influenced neurotransmitter levels. Briefly, the serotonin pathway was modified by CBS overexpression in various brain areas in female mice but not in male mice. The dopamine pathway was modified in brain regions according to sex and age. These results may allow us to better understand the role of the transsulfuration pathway and especially CBS overexpression in the metabolism of biogenic amines and the catecholamine catabolism in persons with trisomy 21.

Insecticide Transfer Efficiency and Lethal Load in Argentine Ants.

Trophallaxis between individual worker ants and the toxicant load in dead and live Argentine ants (Linepithema humile) in colonies exposed to fipronil and hydramethylnon experimental baits were examined using accelerator mass spectrometry (AMS). About 50% of the content of the crop containing trace levels of 14C-sucrose, 14C-hydramethylnon, and 14C-fipronil was shared between single donor and recipient ants. Dead workers and queens contained significantly more hydramethylnon (122.7 and 22.4 amol/µg ant, respectively) than did live workers and queens (96.3 and 10.4 amol/µg ant, respectively). Dead workers had significantly more fipronil (420.3 amol/µg ant) than did live workers (208.5 amol/µg ant), but dead and live queens had equal fipronil levels (59.5 and 54.3 amol/µg ant, respectively). The distribution of fipronil differed within the bodies of dead and live queens; the highest amounts of fipronil were recovered in the thorax of dead queens whereas live queens had the highest levels in the head. Resurgence of polygynous ant colonies treated with hydramethylnon baits may be explained by queen survival resulting from sublethal doses due to a slowing of trophallaxis throughout the colony. Bait strategies and dose levels for controlling insect pests need to be based on the specific toxicant properties and trophic strategies for targeting the entire colony.

Changes in the concentration and relative abundance of alkanes and PAHs from the Deepwater Horizon oiling of coastal marshes.

We determined changes of 28 alkanes and 43 different PAHs in 418 wetland soil samples collected on ten sampling trips to three Louisiana estuaries before and after they were oiled from the 2010 Deepwater Horizon disaster. There was a significant decline in 22 of the 28 alkane analytes (0.42% day(-1)), no change in 6, over 2.5 years. The concentration of five aromatic petroleum hydrocarbons (PAHs) increased (range 0.25-0.70% day(-1)), whereas the total PAH pool did not change. Of these five, naphthalene and C-1-naphthalenes are suggested to be of higher toxicity than the other three because of their relatively higher volatility or solubility. The relative proportions of alkane analytes, but not PAHs, does not yet resemble that in the pre-oiled marshes after 3 years, The trajectories of nine indicators for degradation/weathering were either inconclusive or misleading (alkanes) or confirmed the relatively meager degradation of PAHs.

The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway.

The aryl hydrocarbon receptor (AhR) is commonly described as a transcription factor, which regulates xenobiotic-metabolizing enzymes. Recent studies have suggested that the binding of ligands to the AhR also activates the Src kinase. In this manuscript, we show that the AhR, through the activation of Src, activates focal adhesion kinase (FAK) and promotes integrin clustering. These effects contribute to cell migration. Further, we show that the activation of the AhR increases the interaction of FAK with the metastatic marker, HEF1/NEDD9/CAS-L, and the expression of several integrins. Xenobiotic exposure, thus, may contribute to novel cell-migratory programs.

Novel transglutaminase 1 mutations in patients affected by lamellar ichthyosis.

Lamellar Ichthyosis (LI) is a form of congenital ichthyosis that is caused by mutations in the TGM1 gene that encodes for the transglutaminase 1 (TG1) enzyme. Functional inactivation of TG1 could be due to mutations, deletion or insertions. In this study, we have screened 16 patients affected by LI and found six new mutations: two transition/transversion (R37G, V112A), two nonsense mutations and two putative splice site both leading to a premature stop codon. The mutations are localized in exons 2 (N-terminal domain), 5, 11 (central catalytic domain), and none is located in the two beta-barrel C-terminal domains. In conclusion, this study expands the current knowledge on TGM1 mutation spectrum, increasing the characterization of mutations would provide more accurate prenatal genetic counselling for parents at-risk individuals.

Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity.

Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.

Retrotransposon expression as a defining event of genome reprogramming in fertilized and cloned bovine embryos.

Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

Revealing the dynamics of gene expression during embryonic genome activation and first differentiation in the rabbit embryo with a dedicated array screening.

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.

Amplitude independent RF instantaneous frequency measurement system using photonic Hilbert transform.

A photonic instantaneous frequency measurement system capable of measuring both RF frequency and power simultaneously, is conceived and practically demonstrated. This system employs an RF photonic Hilbert transformer together with low-cost, low-frequency photo-detectors to obtain two orthogonal DC measurements. This system exhibits a frequency range of 1-10 GHz. Wider frequency range can be achieved through integration.

Use of packaged entrees as part of a weight-loss diet in overweight men: an 8-week randomized clinical trial.

AIM: This study assessed the efficacy of a weight-loss diet by using packaged portion-controlled entrees vs. a self-selected diet based on the United States Department of Agriculture Food Guide Pyramid (FGP). METHODS: Sixty healthy overweight men (body mass index (BMI) 26-42 kg/m2; aged 24-60 years) were randomized into two groups for an 8-week intervention. Group E consumed two portion-controlled entrees daily, plus recommended servings from the FGP. Group P consumed a self-selected diet consisting of a recommended number of servings from the FGP. Diets were designed to be isocaloric (1700 kcal) and identical in macronutrient composition (55% carbohydrate, 25% protein and 20% fat). Participants were instructed to make no changes in physical activity levels. Each group was blinded to the protocol of the other group, and received separate diet instructions, but no behavioural or diet counselling. Outcomes included weight, BMI, body composition by dual energy X-ray absorptiometry, waist and hip circumference, blood pressure (BP), fasting blood lipids, glucose, insulin and C-reactive protein. RESULTS: Fifty-one men completed the study. The portion-control group E (n = 25) experienced greater decreases in weight (-7.4 +/- 3.1 vs. -5.1 +/- 4.0 kg), BMI (-2.4 +/- 1.0 vs. -1.6 +/- 1.3 kg/m2), fat mass (-3.6 +/- 1.8 vs. -2.5 +/- 1.8 kg), waist circumference (-6.6 +/- 3.3 vs. -4.3 +/- 2.9 cm) and diastolic BP (-6.0 +/- 7.2 vs. + 0.2 +/- 10.1 mmHg) than group P (n = 26) (p < 0.05). Consumption of a packaged entree diet resulted in greater losses of weight and fat mass, and reduced BP. CONCLUSIONS: Use of packaged entrees as part of a weight-loss diet is an effective means of achieving portion control and enhancing losses of weight and fat mass in overweight men.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms.

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

SSH adequacy to preimplantation mammalian development: scarce specific transcripts cloning despite irregular normalisation.

BACKGROUND: SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. RESULTS: Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts. CONCLUSION: The close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts provides an easy and reliable means to check SSH performance. Despite some cases of irregular normalisation and subtraction failure, we have shown that SSH repeatedly enriches the libraries in very rare, tester-specific transcripts, and can thus be considered as a powerful tool to investigate situations where small amounts of biological material are available, such as during early mammalian development.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells.

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

Efficacy of bifenthrin treatment zones against red imported fire ant.

Exclusion of ants, particularly red imported fire ant, Solenopsis invicta (Buren), from homes, nursing facilities, hospitals, and electrical housings is an important strategy in urban and rural pest control. We conducted a laboratory bioassay to determine the repellency of granular bifenthrin (Talstar: rate 2.087 kg of formulated product/92.88 m2 or 4.6 lb formulated product/1000 feet2 or 4.2 g active ingredient/92.88 m2) to S. invicta foragers. In the field, we compared the efficacy of three widths (0.3, 2.0, and 3.0 m) of granular bifenthrin-treated zones at the rate 2.087 kg of formulated product/92.88 m2 and investigated the survival of individual ants successfully crossing the respective zones. Granular bifenthrin was nonrepellent to fire ant foragers in the laboratory. The 2.0- and 3.0-m treatment zones provided 100% protection for 7 wk after treatment and provided a reduction in the number of ants breaching the treated zone compared with the control for the remaining 9 wk of the study. This level of control may be tolerable for homeowners and is, therefore, considered an effective treatment for 15 wk after treatment. Hospitals, nursing homes, and electrical boxes would have to be treated on a monthly or bimonthly to remain ant free.

An oral bioassay for the toxicity of hydramethylnon to individual workers and queens of Argentine ants, Linepithema humile.

We have developed an oral bioassay to determine the toxicity of hydramethylnon to individual workers and queens of the Argentine ant, Linepithema humile. We fed seven concentrations of hydramethylnon in suspension to individual workers or queens, determined the amount of hydramethylnon ingested and evaluated the individual ants for mortality 14 days later. At concentrations > or = 0.37 g liter-1, the amount of liquid the queens ingested decreased dramatically, indicating that Argentine ant queens may detect hydramethylnon. Significantly larger volumes of the two highest concentrations of the hydramethylnon suspension were ingested by the workers, compared to the lower concentrations, suggesting that hydramethylnon may act as a feeding stimulant for the workers. Worker mortality was higher than queen mortality at the highest concentrations tested. The highest worker mortality resulted when the ants ingested 1.03 micrograms of hydramethylnon per mg of ant tissue. At the highest concentration (1.0 g liter-1) tested, workers ingested almost 12 times as much active ingredient per mg of body weight as did queens, suggesting that, in order to increase mortality of queens, multiple feedings must occur.

Human kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas.

Previous studies indicated that a new member of the human kallikrein (KLK) gene family, KLK4, was expressed in prostate, breast, and endometrial carcinoma cell lines and may have potential as a tumor marker. The aim of this study was to examine the expression of KLK4 in the normal ovary and ovarian tumors of different histology, stage, and differentiation and to determine its association with ovarian tumor progression. Using reverse transcription-PCR, Southern blot, and densitometry analyses, we found the level of KLK4 expression was higher in late stage serous (SER) epithelial-derived ovarian carcinomas than in normal ovaries, mucinous epithelial tumors, and granulosa cell tumors. KLK4 was highly expressed in all of the SER ovarian carcinoma cell lines (eight of eight), SER epithelial carcinomas (11 of 11), and two adenomas, whereas it was expressed at a lower level (or not at all) in normal ovaries (four of six), mucinous epithelial tumors (three of four), endometrioid carcinomas (four of five), clear cell carcinomas (two of three), or granulosa cell tumors (three of six). Of particular interest, KLK4 mRNA variants were detected in SER ovarian carcinoma cell lines and primary cultured ovarian tumor cells, but they were not present in normal ovaries. In situ hybridization analysis showed that KLK4 mRNA transcripts are localized to adenocarcinoma cells of ovarian tumor tissues. Similarly, immunohistochemical staining of ovarian carcinoma sections showed immunoreactivity to KLK4 protein product (hK4) antipeptide antibodies. In addition, intracellular hK4 levels, as detected on Western blot analysis, were induced by 100 nM estrogen treatment of the estrogen receptor positive ovarian carcinoma cell line OVCAR-3, >8-24 h. Our results show that the level of KLK4 expression and expression of KLK4 mRNA variants are associated with progression of ovarian cancer, particularly late stage SER adenocarcinomas. Moreover, hK4 may be a candidate marker for the diagnosis and/or monitoring of ovarian epithelial carcinomas.

Identification and characterization of KLK14, a novel kallikrein serine protease gene located on human chromosome 19q13.4 and expressed in prostate and skeletal muscle.

The kallikreins are a subfamily of serine proteases encoded in human, mouse, and rat by highly conserved tightly clustered multigene families. Here we report the identification and characterization of KLK14, a novel kallikrein gene located within the human kallikrein locus at 19q13.4. KLK14 is approximately 5.4 kb in length spanning seven exons and, by Northern blot analysis, transcribes two alternative transcripts present only in prostate (1.5 kb) and skeletal muscle (1.9 kb). The protein product, K14, predicted to be a 251-amino-acid secreted serine protease with trypsin-like substrate specificity, is translated in vitro with a molecular mass of approximately 31 kDa. In situ hybridization revealed that, in prostate, KLK14 is expressed by both benign and malignant glandular epithelial cells, thus exhibiting an expression pattern similar to that of two other prostatic kallikreins, KLK2 and KLK3, which encode K2 and prostate-specific antigen, respectively.