Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF)

Neuropathic pain concurrent with mood disorder from peripheral nerve injury is a serious clinical problem that significantly affects quality of life. Recent studies have suggested that a lack of brain-derived neurotrophic factor (BDNF) in the limbic system may cause this pain-emotion. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but the role of 4-MC-induced BDNF in pain-emotion is poorly understood. Thus, we assessed the possible involvement of BDNF in brain in depression-like behavior during chronic pain following peripheral nerve injury. In addition, we examined whether intracerebroventricular (i.c.v.) 4-MC prevents chronic pain in rats and produces an antidepressant effect. Sprague-Dawley rats implanted intracerebroventricularly with a PE-10 tube were subjected to chronic constriction injury (CCI). Pain was assessed by a reduction in paw withdrawal latency (PWL) to heat stimuli after CCI. We also used a forced swimming testing (FST; time of immobility, in seconds) from day 14 to day 21 after CCI. Modulation of pain and emotional behavior was performed by injection of PD0325901 (a MEK1/2 inhibitor). 4-MC (100 nM) was continuously administered i.c.v. for 3 days during the period from day 14 to day 21 after CCI. To block analgesic and antidepressant effects, anti-BDNF antibody or K252a (a TrkB receptor inhibitor) was injected in combination with 4-MC. Naloxone was also coadministered to confirm the analgesic effect of 4-MC. During the chronic stage after CCI, the rats showed a sustained decrease in PWL (thermal hyperalgesia) associated with extension of the time of immobility (depression-like behavior). PD0325901 significantly reduced the decrease in PWL and the increased time of immobility after CCI. The decreased PWL and increased time of immobility were also reduced by 4-MC and by treatment with an ERK1/2 inhibitor. These effects of 4-MC i.c.v. were reversed by anti-BDNF and K252a. The analgesic effect of 4-MC i.c.v. was also antagonized by naloxone. Based on these results, we suggest that a lack of BDNF and activation of ERK1/2 in the pain-emotion network in the CNS may be involved in depression-like behavior during chronic pain. 4-MC i.c.v. ameliorates chronic pain and depression-like behavior by producing of BDNF and normalization of ERK1/2 activation. Therefore, enhancement of BDNF may be a new treatment strategy for chronic pain associated with depression.     

Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan

BACKGROUND: Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1beta or glucose. METHODS: Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in "functional" CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA. RESULTS: PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1beta or 25mM D-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation. CONCLUSIONS: The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC-HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.     

Phylogenetic analysis of Lactococcus lactis subspecies based on decoding the sequence of the pepT tripeptidase gene, the pepV dipeptidase gene and 16S rRNA

Tripeptidase (PepT) and dipeptidase (PepV), the enzymes located in the final stage of the intracellular proteolytic system, were demonstrated to be distributed widely in lactic acid bacteria, especially in lactococci. Both the tripeptidase genes (pepT) and dipeptidase genes (pepV) of 15 lactococcal strains consisting of the type and domestic strains were cloned and sequenced using normal and TAIL PCR methods. Amino acid sequences of these enzymes were highly conserved among strains. Evolutionary distance trees based on the sequence of 1239 nucleotides of pepT and 1416 nucleotide of pepV showed a similar cluster as that obtained from the 1499 fragment of the 16S rRNA. Based on this profile, the species Lactococcus lactis is reasonably divided into three subspecies groups, subsp. lactis, cremoris, and hordniae, as in the current classification. Figure of trees from pepT and pepV were essentially identical to each other and slightly more intricate than that from 16S rRNA. The K nuc values obtained from pepT and pepV genes were approximately ten times as high as that from 16S rRNA. Considering these results, phylogenetic analysis based on pepT and pepV genes may aid in a more precise index of classification of L. lactis subspecies. PepT and PepV seem to have evolved in similar directions in lactococci.     

A rapid genotyping method for the vivax malaria transmission-blocking vaccine candidates, Pvs25 and Pvs28

The antigenic diversity observed in many vaccine candidates is one of the difficulties to design effective malaria vaccine. Since it is prerequisite to survey genetic polymorphism of the vaccine candidate antigens for the vaccine development, it is necessary to establish efficient screening method to detect the genetic polymorphism from a large number of samples. Here, we have established efficient polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) method to detect nucleotide diversity of the malaria transmission-blocking vaccine candidates Pvs25 and Pvs28. We can distinguish all 4 haplotypes of Pvs25 by this method. By introducing BsmI-digestion step for Pvs28, we can distinguish 15/16 haplotypes by single electrophoresis. Since this method requires neither sequencing nor radioisotope labeling, it will be easy to transfer the method into a field based high throughput screening of genetic polymorphism.     

Transmission-blocking vaccine of vivax malaria

Malaria remains one of the leading causes of both morbidity and mortality of humans residing in tropical countries. For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and Plasmodium vivax. The genes coding for two potential P. vivax transmission-blocking antigens, Pvs25 and Pvs28, have been cloned. Mice vaccinated with yeast-produced recombinant proteins Pvs25 and Pvs28 adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens. The development of oocysts in mosquitoes was completely inhibited when these antisera were ingested with the P. vivax Salvador (Sal) I strain-infected chimpanzee blood. In a large collection of P. vivax field isolates, we found only 5 nucleotide changes that would result in amino acid substitutions in Pvs25. In contrast, the Pvs28 gene had 22 nucleotide changes that would result in conservative amino acid substitutions. How the antigenic polymorphism of Pvs25 and Pvs28 would affect the efficacy of Sal I based vaccine remains to be elucidated. Clinical trials with Pvs25 and the P. falciparum ortholog Pfs25 are in preparation.     

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.     

The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 via a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103-108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.     

Dietary fat ingestion activates β-endorphin neurons in the hypothalamus

The opioid system regulates food choice, consumption, and reinforcement processes, especially for palatable meals such as fatty food. β-Endorphin is known as an endogenous opioid peptide produced in neurons of the hypothalamus. In this study, we found that Intralipid (fat emulsion) ingestion increased c-fos expression in β-endorphin neurons. However, intragastric infusion of Intralipid only slightly increased c-fos expression 2h after infusion. Further, dissection of glossopharyngeal nerve, innervating posterior tongue taste buds, partially but significantly decreased the Intralipid-induced c-fos expression. These results indicate that mainly the orosensory stimulation from fat may activate β-endorphin neurons, thereby promoting β-endorphin release.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.