Germin isoforms are discrete temporal markers of wheat development. Pseudogermin is a uniquely thermostable water-soluble oligomeric protein in ungerminated embryos and like germin in germinated embryos, it is incorporated into cell walls.

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.

Wheat Ec metallothionein genes. Like mammalian Zn2+ metallothionein genes, wheat Zn2+ metallothionein genes are conspicuously expressed during embryogenesis.

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P-labeled oligonucleotide probes that encoded parts of the partial amino-acid sequence for the Zn-containing Ec protein. Each DNA insert in 11 positives from a screen of 10(5) plaques encoded a 5' untranslated and a 3' untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the Ec protein. The three different mRNA sequences encoded in the cDNA probably correspond to single-copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P-labeled cDNA and gave a single positive in a screen of 5 x 10(5) plaques. A 3.1-kb genomic fragment (gf-3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf-3.1 sequence encodes an intronless mRNA for the Ec protein and contains appreciable amounts of 5' and 3' flanking sequences. In addition to a putative TATA box, two inverted-repeat sequences and one direct-repeat sequence, the 5' flank in gf-3.1 contains a sequence similar to the abscisic-acid-responsive element in other higher-plant genes but does not contain sequences similar to the metal-responsive elements in animal metallothionein genes. Consistent with these findings, RNA blotting shows that accumulation of Ec mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat Ec metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins.

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Night sedation and arthritic pain.

This pilot study highlights the problems of benzodiazepine (BDP) usage in patients attending a rheumatology clinic. Of 127 consecutive patients attending the rheumatology clinic a total of 29% (37) had been taking night sedation for mean duration of 4.1 years. The majority of BDP users (92%) were women. In 78% night sedation was taken for insomnia associated with night pain. We recommend that if BDP is to be prescribed it should be only in selected cases for a short time.

Covalently bonded and adventitious glycans in germin.

Germin was previously shown to contain covalently bonded and adventitious glycans. The object of the present investigation was to characterize the two types of glycan. The presence of N- but not O-glycans in germin is indicated by the biosynthesis of altered forms, including an unglycosylated form of germin when wheat embryos are germinated in the presence of tunicamycin. After treating the doublet of germin pentamers (G and G') from normally germinated embryos with beta-N-acetylglucosaminidase, G is converted to a form that co-migrates with G' during electrophoresis in sodium dodecyl sulfate-polyacrylamide, but G' is unaffected. This suggests that the N-glycans in G contain antennary N-acetylglucosamine but that those in G' do not. This conclusion has been confirmed and elaborated by doubly labeling G and G' in vivo with [3H]glucosamine and [35S]methionine, and by characterizing sugar-labeled glycopeptides from G and G' by gel filtration, before and after their degradation by exoglycosidases. In the context of proven structures for the complex N-glycans in other plant glycoproteins, the findings, when combined with monosaccharide analyses of G and G', permit plausible speculation about the structure of the single N-glycan that is likely present in each G monomer (GlcNAc2(Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc] and G' monomer ((Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc)). The adventitious glycans, which can be removed by phenolic extraction of germin, have a composition similar to that expected for the characteristic hemicelluloses and pectins in monocot cell walls. The possible significance of this finding is discussed in relation to our continuing efforts to define the biochemical involvements of germin. In allied studies, affinity of its N-linked glycans for concanavalin A has been used to concentrate small amounts of germin from large volumes of wheat extract and to fractionate germin from tunicamycin-treated and normally germinated wheat embryos.

Polypeptide structure of germin as deduced from cDNA sequencing.

Synthesis of a relatively rare glycoprotein (germin) signals the onset of growth in germinating wheat embryos. Germin mRNA (1075 nucleotide residues) has an 85-residue 5'-untranslated sequence, a 69-residue sequence that can encode a 23-residue signal-peptide sequence, a 603-residue sequence that can encode a 201-residue mature-protein sequence, and a 318-residue 3'-untranslated sequence that begins with a UAA-terminator codon, ends with a 63-residue polyadenylate tract, and has three polyadenylation (and other, related) signals (AAUAAN etc.). One polyadenylation signal is just 9 nucleotides from the polyadenylation site, the shortest stretch of nucleotides yet found between polyadenylation signal and site in any animal or plant mRNA. The mature-protein coding sequence in germin mRNA contains an unusually high proportion (87%) of G + C in the third positions of its codons. The amino acid sequence of germin does not have extensive internal homologies or repetitions, and it is not characterized by regions of unusually high charge density, as is nucleoplasmin, another water-soluble homopentameric protein with otherwise closely related structural properties. Germin does, however, contain a stretch of 34 uncharged amino acid residues and these may possibly mediate its homopentameric structure and its remarkable resistance to enzymic proteolysis. In view of a possible association of germin with cellular membranes, the most interesting relatedness of the germin sequence to the sequences of other proteins is an 80% homology between a decapeptide sequence in mature germin and a decapeptide sequence in Escherichia coli glycerol-3-phosphate acyltransferase. The relation of germin-gene structure to overall gene regulation during early plant growth is discussed.

Morphometric comparison of synovium from patients with osteoarthritis and rheumatoid arthritis.

Synovium was collected from 15 patients who were undergoing joint surgery. Two groups were defined by clinical diagnosis: patients with primary osteoarthritis (n = 4); and those with rheumatoid arthritis (n = 11). The synovium was studied using histological and morphometric techniques. In agreement with previous studies, no histological features specific for either diagnosis were found. A previously validated morphometric method was used to estimate the cellular density of randomly picked fields within defined areas of synovium. The mean nuclear density of cellularity of comparable areas of synovium was significantly different between these two disease states, but the mean nuclear density between individual representative samples within each clinical group was homogeneous. The morphometric analysis of lymphocyte subsets showed that within the upper synovial region and cellular aggregates in osteoarthritis, the distribution of T cells expressing the CD4 and CD8 antigen was the same. In rheumatoid arthritis CD8 cells predominated in the upper synovial region and CD4 cells in the cellular aggregates. Plasma cells were rarely found in osteoarthritic synovia, but were common in rheumatoid arthritis, with IgG-producing plasma cells predominating. Morphometric studies of representative synovial samples may help to improve histological diagnosis and our understanding of pathological mechanisms.

Representative sample of rheumatoid synovium: a morphometric study.

The synovium from 11 patients with rheumatoid arthritis, who were undergoing joint surgery, was assessed using histological and morphometric techniques. Histological examination confirmed previous reports that the intensity of the cellular reaction varied throughout the synovium, and the morphometric method reflected this variability sensitively. The method was shown to be reproducible and allowed areas of similar cellular density to be defined. From these defined areas a total of 2.5 mm2 of synovium equivalent to 12 fields at x250 required analysis to reflect the variation in the cellular reaction. It would be feasible to collect this amount of material using an arthroscope.

A rheumatoid arthritis B cell subset expresses a phenotype similar to that in chronic lymphocytic leukemia.

An abnormal subpopulation of B cells expressing the T1 antigen, which is normally restricted to T cells, was demonstrated in the peripheral blood of 16 patients with rheumatoid arthritis. This T1+ B cell population accounted for a mean of 19.6% (upper limit 48%) of the circulating B cells and did not correlate with clinical disease activity, rheumatoid factor, or drug treatment. The highest percentage of T1+ B cells found in the blood of 8 patients with connective tissue diseases, including systemic lupus erythematosus, was 5% of the B cells, and for normal controls, it was was 3% of the B cells. As previously reported, we confirmed that the T1+,Ig+ phenotype was a feature of leukemic cells in B-type chronic lymphocytic leukemia. The finding of increased numbers of T1+ B cells in patients with rheumatoid arthritis and those with B-type chronic lymphocytic leukemia raises the possibility that these cells play a role in a spectrum of diseases, including those involving autoimmunity and malignancy.

Crystal deposition in the knee and great toe joints of asymptomatic gout patients.

Crystal deposition in asymptomatic knee and first metatarsophalangeal (MTP) joints has been studied in 31 patients with previously proven gout. All had had clinical gout in their MTP joints but their knee joints had never been the site of acute gout. Knee arthroscopy was performed permitting synovial membrane inspection, photography and biopsy. Crystalline material was seen in 9 knees (28%) and confirmed histologically as monosodium urate (MSU) in 4 (12.5%). Synovial fluid analysis on 26 samples using a polarizing light microscope demonstrated MSU crystals in 4 (12.5%) and calcium pyrophosphate dihydrate (CPPD) in 2 (6%). Fluid aspirated from 27 of the metatarsophalangeal joints revealed MSU crystals in 14 (52%) and no CPPD crystals.

Synthesis and turnover of proteins and mRNA in germinating wheat embryos.

The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early inhibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0--1 h) of postimbibition development, and easily detected during "lag" phase (1--5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition. The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.

Structural integrity of DNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes.

After treatment at a microsomal nuclease concentration too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate, there is little, if any, intact 26 S RNA left in the ribosomes of either wheat germ or rabbit reticulocyte cell-free protein synthesizing extracts. The primary scissions, probably at highly exposed sites in the rRNA of plant and animal ribosomes, produce two fragments which remain complexed until thermal denaturation reveals "hidden breaks." Molecular weights of the fragments are approximately 0.5 x 10(6) and 0.8 x 10(6) in the case of wheat, and 0.4 x 10(6) and 1.3 x 10(6) in the case of rabbit. There is little perceptible degradation of 5 S, 5.8 S, and 18 S rRNA, or of tRNA in the same extracts. Even though limited degradation of 26 S rRNA by a reticulocyte nuclease has been reported to severely impair the translational mechanism in reticulocyte ribosomes, micrococcal nuclease-induced degradation of rRNA, whether limited or extensive, does not seriously impair the ability of reticulocyte lysates to discriminate, by selective polypeptide synthesis, between complex populations of cellular mRNA. In an allied study, it is shown that under conditions well suited to recovery of the 5.8 S/26 S rRNA complex, with its naturally occurring hidden break, 5 S/18 S rRNA complexing is not detectable in the RNA or metabolizing embryos, nor in the RNA from untreated or nuclease-treated protein synthesizing extracts from wheat and rabbit. The significance of this finding is briefly elaborated in relation to the suggestion that 5 S rRNA may interact with the M2(6)A-m2(6)A hairpin near the 3'-end of 18 S rRNA.