IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales.

IS900 PCR for Mycobacterium paratuberculosis was applied to cream, whey, and pellet fractions of centrifuged whole cows' milk. The test and simultaneous control reactions gave correct results for spiked milk and for native milk samples obtained directly from M. paratuberculosis-free, subclinically infected, and clinically infected cows. The test was then applied to units of whole pasteurized cows' milk widely obtained from retail outlets throughout central and southern England from September 1991 to March 1993. With peak periods in January to March and in September to November, when up to 25% of units were affected, an overall 22 of 312 samples (7%) tested positive for M. paratuberculosis. In 18 of the 22 positive samples (81%), the PCR signal segregated to the cream or pellet fractions or both, consistent with the presence of intact mycobacteria. Nine of 18 PCR-positive milk samples (50%) and 6 of 36 PCR-negative milk samples (16%) yielded long-term liquid cultures which tested positive for M. paratuberculosis after 13 to 40 months of incubation, despite overgrowth by other organisms. Taken together with data on the prevalence of M. paratuberculosis infection in herds in the United Kingdom, the known secretion of M. paratuberculosis in milk from subclinically infected animals, and the inability of laboratory conditions simulating pasteurization to ensure the killing of all these slowly growing or unculturable organisms, there is a high risk, particularly at peak times, that residual M. paratuberculosis will be present in retail pasteurized cows' milk in England.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.     

Valve and seta (spine) morphogenesis in the centric diatomChaetoceros peruvianus Brightwell

Summary InChaetoceros peruvianus, the two very long, delicately tapered setae (spine-like processes) of the lower valve curve downwards gently until they are often almost parallel, while those emerging from the upper valve curve sharply downwards until oriented almost in the same direction as the setae of the lower valve. This curvature creates a deep pit between the bases of the upper valve's setae, where they emerge from the valve. In live cells, extension of setae is rapid and very sensitive to disturbance. After cleavage the new silica deposition vesicle (SDV) appears in the centre of the furrow and expands outwards over it. A distinct microtubule centre (MC) appears directly on top of the SDV. Microtubules (MTs) from the MC ensheath the nucleus, and others fan out over the SDV and plasmalemma. A little later, the MC in the lower daughter cell moves off the SDV, and its MTs now appear to mould the plasmalemma/ SDV into the deep pit between the base of the setae. In the upper daughter cell, the MC remains on the SDV. Initiation of setae is first observed as protuberances of bare cytoplasm growing from the sides of the daughter cells, through gaps in the parental valve. Many MTs initially line the plasmalemma of these protuberances as they grow outwards and the SDV also expands over the new surface. As the setae get longer, a unique complex of three organelles appears. Just behind the naked cytoplasm at the tip of the seta, a thin flat layer of fibrous material lines the plasmalemma. This, the first of the complex, is called the thin band. Immediately behind this is the second, a much thicker, denser fibrous band, the thick band. At the front edge of the SDV, 5–6 finger-like outgrowths of silicified wall grow forwards. These are interconnected by the elements of the thick band which thus apparently dictate the polygonal profile of the seta. These also appear to generate the spinules (tiny spines) that adorn the surface of the seta; the spiral pattern of the spinules indicates that this whole complex might differentiate one after the next, in order. Further back from the tip, evenly spaced transverse ribs are formed. These are connected to the third organelle in the complex, the striated band; our interpretation is that the striated band sets up the spacing of the ridges that regularly line the inner surface of the setae. During seta growth, this complex is apparently responsible for controlling the delicate tapering curvature of the very fine silica processes. Since the complex is always seen near the tip of the seta, we conclude that it migrates forwards steadily as the tip grows. While the thin and thick bands could slide continuously over the cell membrane, the striated band must be disassembled and then recycled forward during extension if it is indeed connected to the ridges lining the inside of the setae. We could find no indication that turgor pressure drives extension of the setae, in which event the activity of these organelles is responsible for growth using the justformed silica tube as the base from which extension is generated.     

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.     

Interconversion of the salicylic acid signal and its glucoside in tobacco

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

Spontaneous allelic variant in deafness–blindness gene Ush1g resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1g^bw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1g^bw/bw mice had no substantial retinal degeneration compared with Ush1g^bw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.     

Gonadotropin-releasing hormone in elasmobranch (electric ray, Torpedo marmorata) brain and plasma: chromatographic and immunological evidence for chicken GnRH II and novel molecular forms.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.