Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

Distribution study of orally administered lipoic acid in rat brain tissues.

Lipoic acid (LA), an essential cofactor for mitochondrial enzymes and a natural antioxidant, has been explored for the treatment of Alzheimer's disease. However, lipoic acid distribution in brain has not been investigated via oral dosing in human subjects or animals. Therefore, we aim to investigate the distribution of orally administered LA from systemic circulation into rat brain tissues and understand the transport efficiency of lipoic acid across the blood-brain barrier. Brain and blood samples were obtained from male Lister Hooded rats at pre-defined time points after single and chronic oral dosing of LA at 50 mg/kg. Levels of LA were determined using liquid chromatography tandem mass spectrometry. An equilibrium dialysis method was employed to elucidate LA protein binding in brain and blood tissues. Basal endogenous levels of LA in control rats were found to fluctuate between 0.005 and 0.267 microM in blood and 0-0.024 microM in brain after correction for residual blood volume. Pharmacokinetic profiling demonstrated rapid biphasic elimination of LA in blood and poor distribution into various brain regions with levels ranging from 0.0009 to 0.0072 microM. The in vitro and in vivo LA brain:blood partition ratios were 0.1 and -0.01, respectively. Our results demonstrate for the first time that LA does not cross the blood-brain barrier readily and suggest that the antioxidant effect of LA in brain may not be due to its direct effect in the central nervous system.

SB203580 promotes EGF-stimulated early morphological differentiation in PC12 cell through activating ERK pathway.

MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71-74; Hall-Jackson et al. [1999] Onc. 18: 2047-2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.

Gene suppression by tristetraprolin and release by the p38 pathway.

Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.

Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes.

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, we investigated the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling in vitro and in vivo. We show that calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart were characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrated reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreased p38 phosphorylation and increased MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreased MKP-1 protein levels and increased p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes, which is associated with p38 inactivation.

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity.

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.

Cloning and characterization of RLPK, a novel RSK-related protein kinase.

A novel protein kinase whose activity can be stimulated by mitogen in vivo was cloned and characterized. The cDNA of this gene encodes an 802-amino acid protein (termed RLPK) with the highest homology (37% identity) to the two protein kinase families, p90(RSK) and p70(RSK). Like p90(RSR), but not p70(RSK), RLPK also contains two complete nonidentical protein kinase domains. RLPK mRNA is widely expressed in all human tissues examined and is enriched in the brain, heart, and placenta. In HeLa cells, transiently expressed epitope-tagged RLPK can be strongly induced by epidermal growth factor, serum, and phorbol 12-myristate 13-acetate, but only moderately up-regulated by tumor necrosis factor-alpha and other stress-related stimuli. The activity of RLPK stimulated by epidermal growth factor was not inhibited by several known protein kinase C inhibitors nor by rapamycin, a known specific inhibitor for p70(RSK), but could be inhibited by herbimycin A, a tyrosine kinase inhibitor, and partially inhibited by PD98059 or SB203580, inhibitors for the mitogen-activated protein kinase pathways. Recombinant RLPK possesses high phosphorylation activity toward histone 2B and the S6 peptide, RRRLSSLRA. Although purified recombinant RLPK can be phosphorylated by ERK2 and p38alpha in vitro, its activity is not affected by this phosphorylation. Moreover, the treatment of RLPK with acid phosphatase did not reduce its in vitro kinase activity. These data suggest that RLPK is structurally similar to previously isolated RSKs, but its regulatory mechanism may be distinct from either p70(RSK) or p90(RSK)s.

Regulation of the MEF2 family of transcription factors by p38.

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Molecular cloning and characterization of a human protein kinase that specifically activates c-Jun N-terminal kinase.

The c-Jun N-terminal kinases (JNKs), also called stress-activated protein kinases (SAPKs), belong to the mitogen-activated protein kinase (MAPK) gene super-family. Like all the MAPKs, JNKs are activated through dual phosphorylation of a theronine residue and a tyrosine residue by a dual specificity kinase such as JNKK1/MKK4/SEK1. Here, we report the molecular cloning and characterization of hJNKK2 alpha, a human homolog of the recently reported murine MKK7 alpha. hJNKK2 alpha belongs to the MAPK kinase gene family and is expressed in many adult tissues. It is nearly identical to a recently reported human JNKK2 at the kinase domain but with major differences in both amino- and carboxyl-terminal sequences, suggesting that hJNKK2 alpha may be an alternative spliced form of this kinase. Expression of hJNKK2 alpha, but not its related kinases JNKK1/MKK4/SEK1, MEK1, MKK3, or MKK6, leads to strong activation of JNK in several cell lines. No activation of ERK or p38 kinases was observed with this kinase. An in-vitro kinase assay demonstrated that JNK1 activation by hJNKK2 alpha requires phosphorylation of the theronine and tyrosine residues at positions 183 and 185 in JNK1. Furthermore, hJNKK2 alpha activated the JNK-dependent signal transduction pathway in vivo by induction of c-Jun- and ATF2-mediated gene transcription. In conclusion, we have cloned the human homolog of murine MKK7 alpha, which may be an alternative spliced form of human JNKK2 involved in transducing specific upstream signals to regulate JNK activity in vivo.

PRAK, a novel protein kinase regulated by the p38 MAP kinase.

We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo.

The Gambian National Impregnated Bednet Programme: costs, consequences and net cost-effectiveness.

Clinical trials have indicated that treating mosquito nets with insecticide could be a potentially cost-effective method of preventing malaria. As malaria is one of the most common causes of death in children under five in developing countries, there has been substantial interest in whether such findings can be replicated for a country's control programme in practice. The cost-effectiveness of the Gambian National Insecticide-impregnated Bednet Programme (NIBP), from the viewpoint of providers (government and non-governmental agencies) and the community, has been calculated. Information was collected from existing records, interviews with NIBP personnel, observation and household surveys. Information is provided on the resource use consequences of the NIBP in terms of reduced expenditure on anti-malaria preventive measures, treatment in government health services, household financed treatment and "charity" (burial, funeral and mourning activities), as well as cash income lost as a result of child death. The annual implementation cost of the NIBP was D757,875 (US$91,864), of which 86% was recurrent cost. The estimated number of death averted was 40.56. The net implementation cost-effectiveness ratio per death averted and discounted life years gained were D3884 (US$471) and D260 (US$31.5), respectively. Adding the cost of all mosquito nets would increase the cost-effectiveness ratios by over five times, which is an important consideration for countries with a lower coverage of mosquito nets per capita. It is concluded that insecticide-impregnated mosquito nets are one of the more efficient ways of reducing deaths in children under 10 years in rural Gambia.

The p38 MAP kinase pathway and its biological function.

p38 is a mitogen-activated protein (MAP) kinase with structural and functional characteristics that distinguish it from JNK and ERK MAP kinases. p38 activity is upregulated when cells are exposed to a variety of stimuli including bacterial pathogens, proinflammatory cytokines, certain growth factors, and other forms of environmental stress. By regulating downstream substrates that include protein kinases and transcription factors, p38 participates in transmission, amplification, and diversification of the extracellular signal, initiating several different cellular responses. Studies have revealed that activation of p38 pathway is related to many pathological changes that occur in the course of inflammatory/immunologic and cardiovascular diseases.

Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta.

We have cloned and characterized a new member of the p38 group of mitogen-activated protein kinases here termed p38delta. Sequence comparisons revealed that p38delta is approximately 60% identical to the other three p38 isoforms but only 40-45% to the other mitogen-activated protein kinase family members. It contains the TGY dual phosphorylation site present in all p38 group members and is activated by a group of extracellular stimuli including cytokines and environmental stresses that also activate the other three known p38 isoforms. However, unlike the other p38 isoforms, the kinase activity of p38delta is not blocked by the pyridinyl imidazole, 4-(4-fluorophenyl)-2-2(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (identicalto SB202190). p38delta can be activated by MKK3 and MKK6, known activators of the other isoforms. Nonetheless, in-gel kinase assays provide evidence for additional activators. The data presented herein show that p38delta has many properties that are similar to those of other p38 group members. Nonetheless important differences exist among the four members of the p38 group of enzymes, and thus each may have highly specific, individual contributions to biologic events involving activation of the p38 pathways.

Comparison of two methods for enumerating malaria parasites in thick blood films.

Calculation of parasite densities is important for estimating herd immunity to malaria, and for determining end points in field trials for interventions such as malaria vaccines, impregnated bed nets, and chemosuppression. Two methods of enumeration were compared: method 1, in which 100 consecutive high-power fields (HPFs) are examined, and if they all contain at least one parasite, the number per field is then counted in 10-100 of these fields according to density; and method 2, in which the actual number of parasites present in 100 consecutive fields are counted. The first method significantly underestimates parasite density in samples in which less than all high-power fields are parasite-positive. A correction of method 1 is suggested, which results in a parasite density, which is comparable with that obtained using method 2. The correction factor estimated was 2(-In(1 - p)), where p is the proportion of positive HPFs. The correction factor presented will allow accurate estimate of parasite densities per volume of blood even if only the proportion of parasite-positive high-power fields containing at least one parasite are counted.

Malaria is an important cause of anaemia in primigravidae: evidence from a district hospital in coastal Kenya.

A study was undertaken in order to determine the prevalence and aetiology of anaemia in pregnancy in coastal Kenya, so as to establish locally important causes and enable the development of appropriate intervention strategies. 275 women attending the antenatal clinic at Kilifi district hospital, Kenya, were recruited in November 1993. The prevalence of anaemia (haemoglobin [Hb] < 11 g/dL) was 75.6%, and the prevalence of severe anaemia (Hb < 7g/dL) was 9.8% among all parities; 15.3% of 73 primigravidae were severely anaemic, compared with 7.9% of 202 multigravidae (P = 0.07). In primigravidae, malaria infection (Plasmodium falciparum) was strongly associated with moderate and severe anaemia (chi 2 test for trend, P = 0.003). Severe anaemia was more than twice as common in women with peripheral parasitaemia as in those who were aparasitaemic, and parasitaemia was associated with a 2.2g/dL decrease in mean haemoglobin level (P < 0.001). In multigravidae, iron deficiency and hookworm infection were the dominant risk factors for anaemia. Folate deficiency and human immunodeficiency virus infection were not strongly associated with anaemia. It is suggested that an intervention that can effectively reduce malaria infection in primigravidae could have a major impact on the health of these women and their infants.

cDNA cloning of rasp-1, a novel gene encoding a plasma protein associated with liver regeneration.

We constructed a cDNA library using mRNA isolated from liver 48 hr after hepatectomy (HX) and screened it by differential hybridization using cDNA from normal and regenerating rat liver. We isolated one clone termed regeneration-associated serpin-1 (rasp-1) that was expressed in normal liver but was upregulated approximately 3-4 fold by 48 hr after HX. DNA sequence analysis of rasp-1 indicated that it encoded a novel 436 amino acid secreted protein. Moderate homology was found with several members of the serpin family of serine-protease inhibitors. The 1.7 kb raps-1 mRNA was highly expressed in liver but not in brain, heart, kidney, lung, testis or spleen. We also found the RASP-1 protein in normal and HX rat plasma using a polyclonal antibody generated against a deduced peptide of rasp-1. Rasp-1 may encode a novel serine-protease inhibitor associated with liver regeneration.

Insecticide-treated bednets reduce mortality and severe morbidity from malaria among children on the Kenyan coast.

New tools to prevent malaria morbidity and mortality are needed to improve child survival in sub-Saharan Africa. Insecticide treated bednets (ITBN) have been shown, in one setting (The Gambia, West Africa), to reduce childhood mortality. To assess the impact of ITBN on child survival under different epidemiological and cultural conditions we conducted a community randomized, controlled trial of permethrin treated bednets (0.5 g/m2) among a rural population on the Kenyan Coast. Between 1991 and 1993 continuous community-based demographic surveillance linked to hospital-based in-patient surveillance identified all mortality and severe malaria morbidity events during a 2-year period among a population of over 11000 children under 5 years of age. In July 1993, 28 randomly selected communities were issued ITBN, instructed in their use and the nets re-impregnated every 6 months. The remaining 28 communities served as contemporaneous controls for the following 2 years, during which continuous demographic and hospital surveillance was maintained until the end of July 1995. The introduction of ITBN led to significant reductions in childhood mortality (PE 33%, CI 7-51%) and severe, life-threatening malaria among children aged 1-59 months (PE 44%, CI 19-62). These findings confirm the value of ITBN in improving child survival and provide the first evidence of their specific role in reducing severe morbidity from malaria.

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.