Evidence that Griscelli syndrome with neurological involvement is caused by mutations in RAB27A,not MYO5A

Griscelli syndrome (GS), a rare autosomal recessive disorder, is characterized by partial albinism, along with immunologic abnormalities or severe neurological impairment or both. Mutations in one of two different genes on chromosome 15q can cause the different subtypes of GS. Most patients with GS display the hemophagocytic syndrome and have mutations in RAB27A, which codes for a small GTPase. Two patients with neurological involvement have mutations in MYO5A, which codes for an actin-based molecular motor. The RAB27A and MYO5A gene products interact with each other and function in vesicle trafficking. We report the molecular basis of GS in a Muslim Arab kindred whose members have extremely variable neurological involvement, along with the hemophagocytic syndrome and immunologic abnormalities. The patients have normal MYO5A genes but exhibit a homozygous 67.5-kb deletion that eliminates RAB27A mRNA and immunocytofluorescence-detectable protein. We also describe the molecular organization of RAB27A and a multiplex polymerase chain reaction assay for the founder deletion in this kindred. Finally, we propose that all patients with GS have RAB27A mutations and immunologic abnormalities that sometimes result in secondary neurological involvement. The two patients described elsewhere who have MYO5A mutations and neurological complications but no immunologic defects may not have GS but instead may have Elejalde syndrome, a condition characterized by mild hypopigmentation and severe, primary neurological abnormalities.     

A spectrophotometric assay for allicin,alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.     

Intracellular ion and organic solute concentrations of the extremely halophilic bacterium <Emphasis Type="Italic">Salinibacter ruber</Emphasis>

Salinibacter ruber is a red obligatory aerobic chemoorganotrophic extremely halophilic Bacterium, related to the order Cytophagales. It was isolated from saltern crystallizer ponds, and requires at least 150 g l(-1) salt for growth. The cells have an extremely high potassium content, the ratio K(+)/protein being in the same range as in halophilic Archaea of the order Halobacteriales. X-ray microanalysis in the electron microscope of cells grown in medium of 250 g l(-1) salt confirmed the high intracellular K(+)concentrations, and showed intracellular chloride to be about as high as the cation concentrations within the cells. A search for intracellular organic osmotic solutes, using (13)C-NMR and HPLC techniques, showed glutamate, glycine betaine, and N-alpha-acetyllysine to be present in low concentrations only, contributing very little to the overall osmotic balance. The results presented suggest that the extremely halophilic Bacterium Salinibacteruses a similar mode of haloadaptation to that of the Archaea of the order Halobacteriales, and does not accumulate organic osmotic solutes such as are used by all other known halophilic and halotolerant aerobic Bacteria.     

Presence of two novel cardiolipins in the halophilic archaeal community in the crystallizer brines from the salterns of Margherita di Savoia (Italy) and Eilat (Israel)

Two novel cardiolipin derivatives were recently detected in Halobacterium salinarum, namely an archaeal analog of bisphosphatidylglycerol (BPG) and a glycocardiolipin (GlyC). GlyC was found to be tightly bound to bacteriorhodopsin. To obtain information on the presence and distribution of these archaeal cardiolipins, we have analyzed the lipids extracted from the crystallizer ponds of the salterns of Margherita di Savoia (Italy) and Eilat (Israel) and from cultures of representative species of the Halobacteriaceae by electrospray ionization mass spectrometry. BPG was present as a minor lipid component in the lipids extracted from the biomass of the Margherita di Savoia and the Eilat salterns, while GlyC was detected only in the extract of the biomass of Margherita di Savoia. Both compounds were enriched in the membrane fraction obtained by dialysis of the cells against distilled water. We detected BPG in all members of the Halobacteriaceae tested, but GlyC has so far been found only in the genera Halobacterium and Haloarcula. A sulfated diglycosyl diether was the major glycolipid detected in the biomass of both salterns.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, U.S.A., and Eilat, Israel

The whole community pigments and lipids have been examined during a 5-year period in two commercial solar salterns located in the United States and in Israel. There were significant differences in the complexity of the lipid and pigment patterns within the California saltern system, and these differences were not consistent over the sampling period despite examination of ponds with the same salinity. The solar saltern system in Eilat, Israel, showed greater consistency during this sampling period and compared directly with previous studies. The complexity of the saltern in Newark, California, could be explained on the basis of the prevailing weather conditions (cooler and more rainfall) and the nutrient-enriched source water. The Eilat saltern, however, has an oligotrophic water source and has a considerably warmer and drier climate. This difference resulted in more diverse and more complex pigment and lipid patterns and presumably microbial populations in the Newark, California, plant than in the saltern in Eilat, Israel. Received: December 10, 1999 / Accepted: April 6, 2000     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

Differential regulation of MMPs and matrix assembly in chicken and turkey growth-plate chondrocytes

Matrix metalloproteinases (MMPs) play a crucial role in growth-plate vascularization and ossification by processes involving proteolytic cleavage and remodeling of the extracellular matrix (ECM). Their regulation in the growth plate is crucial for normal vs. impaired matrix assembly. Tibial dyschondroplasia (TD), a prevalent skeletal abnormality in avian species, is characterized by the formation of a nonvascularized, nonmineralized plaque in the growth plate. Here, we show differential regulation of MMPs in cultured chondrocytes from chickens and turkeys; retinoic acid (RA) elevated MMP-2 activity in both species, but only in chicken did it induce MMP-9 activity. In contrast, phorbol 12-myristate 13-acetate (PMA) treatment induced MMP-9 activity in turkey chondrocytes but not in those of chicken. Moreover, we found different developmental patterns of TD in chickens and turkeys in-vivo as lower concentrations of, and shorter exposure to thiram were required in chicken than in turkey for TD induction. Growth-plate cartilage taken from thiram-induced lesions had lower gelatinolytic and caseinolytic activities compared with normal cartilage. Likewise, thiram reduced MMP-2 and MMP-13 activity in both chicken and turkey chondrocytes in vitro, although 10-fold higher concentrations were required for this effect in the latter. Finally, the combined treatments of RA or PMA with thiram induced MMP-9 activity in turkey but not in chicken chondrocytes. Furthermore, RA combined with thiram synergistically upregulated its activity in turkey but not chicken chondrocytes. Taken together, these results suggest that mechanisms of MMP regulation differ in the growth plates of these closely related avian species, resulting in altered matrix assembly as exemplified by TD development.     

Extremely halophilic Archaea from Tuz Lake,Turkey, and the adjacent Kaldirim and Kayacik salterns

Tuz Lake is a hypersaline lake located in Central Anatolia, Turkey. The lake and its salterns, Kaldirim and Kayacik, are the major sources of solar salt for industrial applications in Turkey, especially in the food and leather industries. Use of the crude solar salt often results in microbial deterioration of the products. We therefore initiated a thorough characterization of the microbial communities in Tuz Lake and its adjacent salterns, and we present here the results of investigations on diversity of extremely halophilic Archaea. Twenty-seven colonies of aerobic red or pink Archaea (family Halobacteriaceae) were selected according to colony shape, size, consistency and pigmentation, and characterized according to their phenotypic characteristics, polar lipid contents, and antibiotic sensitivities. Furthermore, 16S rRNA genes of the isolates were screened by DGGE analysis and partially sequenced. Phylogenetic analysis showed that most isolates belonged to the genera Haloarcula, Halorubrum and Halobacterium. Haloarcula was found to be dominant both in Tuz Lake and in the saltern samples. Halorubrum species were isolated from Tuz Lake and from the Kaldirim saltern, and Halobacterium species were recovered from Tuz Lake and from the Kayacik saltern. All strains showed various activities of hydrolytic enzymes (proteases, amylases, cellulases, and others), activities which are responsible for the detrimental effects of the crude salt in food and leather products.     

Expression pattern of prokineticin 1 and its receptors in bovine ovaries during the estrous cycle: involvement in corpus luteum regression and follicular atresia

Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.