Calcium montmorillonite clay reduces urinary biomarkers of fumonisin B1 exposure in rats and humans.

Fumonisin B1 (FB1) is often a co-contaminant with aflatoxin (AF) in grains and may enhance AF's carcinogenicity by acting as a cancer promoter. Calcium montmorillonite (i.e. NovaSil, NS) is a possible dietary intervention to help decrease chronic aflatoxin exposure where populations are at risk. Previous studies show that an oral dose of NS clay was able to reduce AF exposure in a Ghanaian population. In vitro analyses from our laboratory indicated that FB1 (like aflatoxin) could also be sorbed onto the surfaces of NS. Hence, our objectives were to evaluate the efficacy of NS clay to reduce urinary FB1 in a rodent model and then in a human population highly exposed to AF. In the rodent model, male Fisher rats were randomly assigned to either FB1 control, FB1 + 2% NS or absolute control group. FB1 alone or with clay was given as a single dose by gavage. For the human trial, participants received NS (1.5 or 3 g day⁻¹) or placebo (1.5 g day⁻¹) for 3 months. Urines from weeks 8 and 10 were collected from the study participants for analysis. In rats, NS significantly reduced urinary FB1 biomarker by 20% in 24 h and 50% after 48 h compared to controls. In the humans, 56% of the urine samples analysed (n = 186) had detectable levels of FB1. Median urinary FB1 levels were significantly (p < 0.05) decreased by >90% in the high dose NS group (3 g day⁻¹) compared to the placebo. This work indicates that our study participants in Ghana were exposed to FB1 (in addition to AFs) from the diet. Moreover, earlier studies have shown conclusively that NS reduces the bioavailability of AF and the findings from this study suggest that NS clay also reduces the bioavailability FB1. This is important since AF is a proven dietary risk factor for hepatocellular carcinoma (HCC) in humans and FB1 is suspected to be a dietary risk factor for HCC and oesophageal cancer in humans.

Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.

The multifunctional 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala; and are associated with the regulation of diuresis in a variety of species of insects. We previously reported the functional expression of a southern cattle tick (Boophilus microplus) G protein-coupled receptor that is activated by insect kinins. Four different stereochemical variants of each of the 4-aminopyroglutamic acid (APy) and tetrazole moieties, mimics of a cis-peptide bond, type VI beta-turn in insect kinins were now evaluated on the expressed tick receptor using a calcium bioluminescence plate assay. This study represents the first investigation of the interaction of restricted-conformation analogs incorporating components that mimic specific conformations and/or peptide bond orientations in an expressed arthropod neuropeptide receptor. Analog Ac-RF[APy]WGa (2R,4S) was at least 10-fold more active than the other analogs, thus identifying the optimal stereochemistry for tick receptor interaction. The optimal stereochemistry for the tetrazole insect kinin analogs in the tick receptor assay was identified as (D,L). The APy is superior to the tetrazole as a scaffold for the design of mimetic insect kinin analogs. These biostable analogs provide new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin regulated processes.

Migration of a Kirschner wire used in the fixation of a subcapital humeral fracture, causing cardiac tamponade: case report and review of literature.

Metallic pins and wires are frequently used for fixation of fractures and dislocations. Migration is one of the potential complications of such fixation methods. Usually, migration of the pins causes only minor complications, but if the device migrates to a vital cavity, serious damage and even death may ensue. The shoulder girdle is one of the areas in which pins and wires are mostly used, the humeral neck fractures being one of them. We report a case in which a Kirschner wire migrated from a subcapital humeral fracture site into the aorta and pericardium, causing sudden cardiac tamponade and death.

Cocaine, but not amphetamine, short term treatment elevates the density of rat brain vesicular monoamine transporter 2.

We compared the effect of 5 days D-amphetamine (5 mg/kg/day i.p.) and cocaine (15 mg/kg/day i.p.) administration on the vesicular monoamine transporter 2 (VMAT2) density in rat brain. VMAT2 expression was assessed by [(3)H]dihydrotetrabenazine high affinity binding. Cocaine administration led to significant increases in VMAT2 density in both prefrontal cortex (+40%, p < 0.01) and striatum (+23%, p < 0.05), while amphetamine did not affect VMAT2 expression. The upregulation of VMAT2 may serve as compensatory mechanism aimed to enhance the vesicular monoamine storage capacity.

Insect satiety: sulfakinin localization and the effect of drosulfakinin on protein and carbohydrate ingestion in the blow fly, Phormia regina (Diptera: Calliphoridae).

Sulfakinins, which are satiety factors in invertebrates, have previously been shown to inhibit feeding in the German cockroach and desert locust. This study examines the occurrence of sulfakinin immunoreactivity and the role of sulfakinin as a feeding satiety factor in the black blow fly, Phormia regina. Specifically, this study examines the effect of sulfakinin on two of the blow fly's nutrient requirements (i.e., carbohydrates and proteins). We observed sulfakinin immunoreactive cells in the brains of both male and female flies. We found that drosulfakinin I (DrmSKI, FDDY[SO(3)H]GHMRFa) significantly inhibited carbohydrate feeding by 44% at the most effective dose (10 nmol) in female flies. Statistically, there was no significant effect on males; however, injections of 10 nmol DrmSKI reduced carbohydrate feeding by 34% compared to the sham. Drosulfakinin had no effect on protein feeding and no significant inhibition was detected in females or males. The results of this study lend further support to the idea that carbohydrate and protein feeding are regulated by separate control mechanisms, especially in Calliphoridae.

Multidisciplinary forensic approach--investigation of a neonaticide and alleged concomitant rape.

In the case presented, a young woman claimed to have spontaneously aborted a 30-weeks old fetus conceived after being raped by her cousin. The police investigation was centred on a probable neonaticide, mitigated by a possible rape. In Israel, the crime of rape carries a heavy punishment similar to that of first degree murder. Thus the implementation of the most recent scientific forensic techniques is of paramount importance in charging or clearing a suspected rapist. The skeletonized fetal remains were found four months after the reputed abortion. The alleged neonaticide was supported by the results of the forensic clinical and anthropological examinations, while the rape accusation was dismissed by the results of the molecular biology identification of the skeletonized fetal remains. The aim of this report is to encourage the forensic community to apply a multi-disciplinary approach, thus maximizing the decision making competence of the courts of law.

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.

A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.

Penile lesions -- reinforcing the case against suspects of sexual assault.

In most cases of sexual assault, even following long term abuse, genital and extra-genital injuries may be very scarce or literally not found. Though the forensic experts customary illustrate to the court that the absence of physical trauma does not rule out that the assault had taken place, more substantial medical evidence can be helpful in pursuing a conviction. We present three cases examined in the National Center of Forensic Medicine in Tel-Aviv in which information obtained during questioning of the victims concerning genital pathology of the assailant was later verified through examination of the suspects. Such data may provide key evidence to get an indictment when the assault is perpetrated by a stranger or a person who should not have an intimate relationship with the victim. Therefore, acquiring information from the victim concerning any pathology of the assailant's genitalia should be part of the routine questionnaire form of victims of suspected sexual assault or abuse as findings of this sort may become paramount in conviction of perpetrators.

Functional analysis of a G protein-coupled receptor from the southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.

The myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis (Mollusca) has been the only previously identified myokinin receptor. We had cloned a G protein-coupled receptor (AF228521) from the tick Boophilus microplus (Arthropoda: Acari), 40% identical to the lymnokinin receptor, that we have now expressed in CHO-K1 cells. Myokinins at nanomolar concentrations induced intracellular calcium release, as measured by fluorescent cytometry and the receptor coupled to a pertussis toxin-insensitive G protein. Absence of extracellular calcium did not inhibit the fluorescence response, indicating that intracellular stores were sufficient for the initial response. Control cells only transfected with vector did not respond. We conclude that the tick receptor is the first myokinin receptor to be cloned from an arthropod.

Isolation, identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei.

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.

In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.).

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.

Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae).

Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 degrees C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 A[1] WP-2, LPK and 434 [phi2] WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (X=Asn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the C-terminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut.

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

The Jeremiah Metzger Lecture. Hypercoagulable states: challenges and opportunities.

Thromboregulatory physiology is essentially a function of the blood vessel wall. Constitutive endothelial cell activities maintain blood fluidity by down regulating the initiation as well as, the propagation of blood coagulation. The major systems involved include: the Protein C, TFPI, plasmin generating and antithrombin pathways, all of which are focused on the cell membrane. Altered regulation of these endothelial functions forms the basis of the pathophysiologic events associated with the inherited primary hypercoagulable states. Secondary hypercoagulable syndromes occurring in various clinical states with conversion to a vascular thrombogenic phenotype reflect non constitutive activated endothelial cell functions with concomitant down regulation of the constitutive anticoagulant surface activity. So called idiopathic clinical thrombosis in most circumstances represents multi hit events in which specific genetic abnormalities or polymorphisms together with specific acquired alterations in geographically distinct endothelial cell beds culminate in a recognizable coagulation phenotype.

Histomorphometric analysis of the normal adult patella.

The patellae of 6 male and 2 female, 40 to 70 year-old individuals, who were healthy at the time of their violent death, were assessed by computer-assisted image analysis. The means of the bone density (percentage of bone in the respective field of interest) ranged from approximately 20% to approximately 30% in the central spongiotic zones, from approximately 40% to approximately 80% in the superior and inferior peripheral zones, and approximately 40% to approximately 60% in the subchondral zone. Bone densities were greatest in the lateral parts of the subchondral and spongiotic territories. The bony trabeculae were haphazardly distributed in the central spongiotic zones. They were commonly oriented vertically or parallel to the surface of the patella in the peripheral and subchondral zones. In conclusion, the histomorphometric data presented validate the rationale of reaming the articular aspect of the patella into a dome-shaped configuration with preservation of a circumferential bony bulwark in the preparation for the implantation of a thick polyethylene-based component with a concave undersurface.

Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs.

In this brief overview we give the historical background on the discovery of myostimulatory neuropeptides in cockroaches. Related peptides were later found in other insect groups as well. We summarize the current knowledge on primary structures, localization, physiological and pharmacological effects of the different cockroach neuropeptides, including kinins, sulfakinins, pyrokinins, tachykinin-related peptides, periviscerokinins, corazonin, and proctolin. In addition, we briefly comment on the development of mimetic pseudopeptide analogs in the context of their possible use in insect pest management.

Newly discovered functions for some myotropic neuropeptides in locusts.

The field of neuropeptide research in insects during the past twenty years can be characterized by the enormous number of peptides that have been identified. In the locusts, Locusta migratoria and Schistocerca gregaria only, structural information is now available for more than 60 peptides. Quite a number of these peptides were isolated on the basis of their effect on visceral muscle contraction in vitro. A very limited number of reports describe the 'in vivo' function of a myotropic neuropeptide. Moreover, for most of the brain neuropeptides, we ignore whether they have a hormonal function. In this paper, we describe the recently discovered in vivo effects of some of the myotropic peptides, identified in locusts in the past decade. Schistocerca-neuropeptide F accelerates egg development; locustasulfakinin inhibits food intake and [His(7)]-corazonin induces body color pigmentation.

Comparative topical pheromonotropic activity of insect pyrokinin/PBAN amphiphilic analogs incorporating different fatty and/or cholic acid components.

This study presents a comparison of the topical pheromonotropic activity in the tobacco budworm moth of a series of amphiphilic pseudopeptide analogs of the insect pyrokinin/PBAN peptide class incorporating fatty acids of varying chain lengths. While the C16 analog fails to penetrate the moth cuticle, and the C12 only moderately so, shorter chain analogs transmigrate the moth cuticle readily with decreasing cuticle-retention properties. A cholic acid analog topically induces twice the maximal pheromone titer of injected native hormone. From a pest management perspective, these non-aromatic hydrophobic components are expected to be more environmentally benign than benzenoid components previously used in topical insect peptide analogs.

Synthesis of 3-(12-hydroxy-p-carboranyl)propionic acid, a hydrophobic, N-terminal tyrosine-mimetic for peptides.

A new, p-carborane containing analog of tyrosine, namely 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran(12)-12-yl]propionic acid was prepared from p-carborane in five steps involving hydroxypropylation of O-protected 1-hydroxy-p-carborane as the key transformation. The simple tyrosine mimetic can function as a hydrophobic surrogate for an N-terminal tyrosine residue in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs.