<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Micropropagation of Gagnepainia godefroyi K Schum and Gagnepainia thoreliana (Baill) K Schum — Rare Medicinal Plants of Thailand

A micropropagation protocol for Gagnepainia godefroyi K Schum and G thoreliana (Baill) K Schum, two rare medicinal plants of Thailand, has been developed using terminal buds. After a total of twenty weeks of culture (4 weeks on MS added with 36.33 μM TDZ and 16 weeks on PGR-free MS), 28.77 and 13.50 shoots/explant were obtained, respectively. Rooting was spontaneous and regenerated plants were successfully transplanted to soil.     

Cysteine-rich secretory protein 2 binds to mitogen-activated protein kinase kinase kinase 11 in mouse sperm

Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an approximately 60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.     

Use of Rhodopseudomonas palustris P1 stimulated growth by fermented pineapple extract to treat latex rubber sheet wastewater to obtain single cell protein

Latex rubber sheet wastewater (non sterile wastewater: RAW) was treated efficiently using a stimulated Rhodopseudomonas palustris P1 inoculum with added fermented pineapple extract (FPE) under microaerobic light conditions. Optimization of wastewater treatment conditions using a central composite design (CCD) found that a 3 % stimulated P1 inoculum with 0.9 % added FPE and a 4-day retention time (RT) were the most suitable conditions. Calculations from CCD experiments predicted that a chemical oxygen demand (COD) of 3,005 mg/L could be 98 % removed, together with 79 % of suspended solids (SS) and 72 % of total sulfide (TtS). No H₂S was detected, production costs were low and single cell protein (SCP) was a by-product. The results of the verification test had an error of only 4–8 % and confirmed removal of COD (initial COD 2,742 mg/L), SS and TtS at 94 %, 75 % and 66 %, respectively. These values were less than the best set obtained from the CCD experiment (2 % stimulated P1 inoculum, 0.75 % FPE and 4 days RT); upon repeating, this set could reduce 96 % of the COD, 78 % SS and 71 % TtS. The treated wastewater met the standard guidelines for irrigation use and no H₂S was detected. The biomass obtaining after wastewater treatment from the best set consisted mostly of R. palustris P1; the biomass of this set had 65 % protein, 3 % fat, 8 % carbohydrate, 14 % ash and 10 % moisture. The results demonstrated that an inoculum of stimulated P1 grew well in RAW supplemented with FPE and could be considered to be an appropriate technology for effectively treating wastewater, with SCP as a by-product.     

The unusual canangafruticosides A-E: Five monoterpene glucosides, two monoterpenes and a monoterpene glucoside diester of the aryldihydronaphthalene lignan dicarboxylic acid from leaves of Cananga odorata var. fruticosa

From the leaves of Cananga odorata var. fruticosa, five unusual monoterpene glucosides, named canangafruticosides A-E (1-5), along with two unusual non-glucosidic monoterpenes (6, 7) were isolated. An aryldihydronaphthalene-type lignan dicarboxylate (8) was also isolated, with two moles of canangafruticoside A (1) on its ester moiety. This lignan also showed strong blue fluorescence emission under basic conditions. The structures of these compounds were elucidated by means of spectroscopic methods, with their absolute configurations determined by application of the modified Mosher’s method to a compound chemically derived from canangafruticoside E.     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein–protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.     

Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments

We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.     

Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.