Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production.

Intestinal microbiota are critical for health with changes associated with diverse human diseases. Research suggests that altered intestinal microbiota can profoundly affect brain function. However, whether altering brain function directly affects the microbiota is unknown. Since it is currently unclear how brain injury induces clinical complications such as infections or paralytic ileus, key contributors to prolonged hospitalization and death post-stroke, we tested in mice the hypothesis that brain damage induced changes in the intestinal microbiota. Experimental stroke altered the composition of caecal microbiota, with specific changes in Peptococcaceae and Prevotellaceae correlating with the extent of injury. These effects are mediated by noradrenaline release from the autonomic nervous system with altered caecal mucoprotein production and goblet cell numbers. Traumatic brain injury also caused changes in the gut microbiota, confirming brain injury effects gut microbiota. Changes in intestinal microbiota after brain injury may affect recovery and treatment of patients should appreciate such changes.

Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.

The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.

RNase cleavage-based methods for mutation/SNP detection, past and present.

Mutation detection based on ribonuclease cleavage of basepair mismatches in single-stranded RNA probes hybridized to DNA targets was first described over 15 years ago. The original methods relied on RNase A for mismatch cleavage; however, this enzyme fails to cleave many mismatches and has other drawbacks. More recently, a new method for RNase-cleavage-based mutation scanning has been developed, which takes advantage of the ability of RNase 1 and RNase T1 to cleave mismatches in duplex RNA targets, when these enzymes are used in conjunction with nucleic acid intercalating dyes. The method, called NIRCA, is relatively low-cost in terms of materials and equipment required. It is being used to detect mutations and SNPs in a wide variety of genes involved in human genetic disease and cancer, as well as in disease-related viral and bacterial genes. This review describes historical and recently developed RNase cleavage-based methods for mutation/SNP scanning.

Detection of mutations by RNase cleavage.

Ribonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA-DNA or RNA-RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of (32)P-labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A Support Protocol explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.

Discreteness and interactivity in spoken word production.

Five theories of spoken word production that differ along the discreteness-interactivity dimension are evaluated. Specifically examined is the role that cascading activation, feedback, seriality, and interaction domains play in accounting for a set of fundamental observations derived from patterns of speech errors produced by normal and brain-damaged individuals. After reviewing the evidence from normal speech errors, case studies of 3 brain-damaged individuals with acquired naming deficits are presented. The patterns these individuals exhibit provide important constraints on theories of spoken naming. With the help of computer simulations of the 5 theories, the authors evaluate the extent to which the error patterns predicted by each theory conform with the empirical facts. The results support a theory of spoken word production that, although interactive, places important restrictions on the extent and locus of interactivity.

Effect of pH on hen egg white lysozyme production and evolution of a recombinant strain of Aspergillus niger.

An Aspergillus niger strain (B1) transformed to produce mature hen egg white lysozyme (HEWL) from a glucoamylase fusion protein under control of the A. niger glucoamylase promoter was grown in glucose-limited chemostat culture at a dilution rate of 0.07 h-1 at various pH values. Maximum HEWL production (9.3 mg g-1; specific production rate = 0.65 mg g-1 per h) was obtained at pH 4.5. However, in chemostat culture, HEWL production was not stable at any pH tested. After 240 h in steady state, specific production decreased to only 0.03 +/- 0.01 and 0.24 +/- 0.02 mg g-1 per h at pH 6.5 and 4.5, respectively. Some isolates removed from the chemostat cultures had lost copies of the HEWL gene and when grown in shake flask cultures all of the isolates produced less HEWL than the parental strain. Morphological mutants with similar phenotypes were isolated at all pHs, but their rate of increase in the population was pH dependent, with cultures at low pH (< 4.5) being more morphologically stable than cultures at high (> 4.5) pH. The selective advantage of these mutants was also generally dependent on pH. Both yellow pigment producing mutants and brown sporulation mutants had higher selective advantages over the parental strain at high than at low pH, regardless of the pH at which they were isolated. However, the selective advantage of densely sporulating mutants was independent of pH.

NIRCA: a rapid robust method for screening for unknown point mutations.

We describe a method for screening for dispersed point mutations, based on the observation that RNase frequently cleaves both strands of base pair mismatches in duplex RNA targets. The mismatched substrates are generated by in vitro transcription of wild-type and mutant templates amplified by the PCR or reverse transcription (RT)-PCR; bacteriophage promoters are incorporated into the PCR primers to permit both strands of the products to be transcribed into RNA. Complementary wild-type and mutant transcripts are hybridized and treated with RNase, and the cleavage products are separated on agarose gels and detected by visualization of the ethidium-stained sample under UV light. The method is thus non-isotopic, and since the cleavage products remain double-stranded during analysis, the labor-intensive RNase inactivation steps required in the original procedure can be eliminated. Also, nonspecific background cleavage is reduced so that longer target regions (1 kb) can be screened in a single step. The Non-Isotopic RNase Cleavage Assay (NIRCA) achieved a detection rate of 88%-90% in blind studies in a Factor IX model system, and it was also used to detect unknown p53 mutations in breast tumor samples. NIRCA provides a rapid method for sensitive, non-isotopic, high-throughput genetic screening.

Analysis of the effect of a strain-specific alteration in the upstream octamer region on transcription of a mouse V kappa Ser light chain gene.

Previous studies had shown that several phenotypic markers expressed by several strains of mice (C58, AKR, PL, RF) involve a light chain group, called V kappa Ser, encoded by a single germ-line gene (Igk-VSera). Most other inbred strains (e.g., BALB/c) contain an allele (Igk-VSerb) which differs from Igk-VSera both in coding regions and in an upstream octamer region known to be important for transcription. Since no evidence for expression of the Igk-VSerb gene product has been observed, experiments were undertaken to determine whether the alteration in the regulatory octamer region of BALB/c might have rendered it defective for transcription. The upstream octamer-containing region of a cloned functional V kappa Ser gene expressed by the C.C58 myeloma M75 was replaced by the corresponding region from BALB/c or deleted entirely. Constructs were transfected into J558L cells and quantity of transcription and site of transcription initiation were compared. Results suggest that the BALB/c octamer (CTTTGCGT), which differs at two of eight nucleotides from the consensus octamer sequence (ATTTGCAT), is fully functional in transcription initiation. This is consistent with results of S1 nuclease protection experiments which indicate the presence of small amounts of correctly initiated V kappa Ser-related RNA in BALB/c spleen.

Structural differences in a single gene encoding the V kappa Ser group of light chains explain the existence of two mouse light-chain genetic markers.

Two phenotypic markers of mouse immunoglobulin kappa light chains, the IB-peptide marker and the Ef1a isoelectric focusing marker, are expressed by the C58/J, AKR/J, RF/J, and PL/J strains (called expressor strains) but not by BALB/c and most inbred strains. Expression is linked to the kappa light-chain locus and the Lyt-2/Lyt-3 genes on chromosome 6. Light chains bearing these markers belong to a group of variable region kappa chain (V kappa) regions called V kappa Ser, which has a serine amino terminus and a framework 1 region not observed to date among BALB/c light chains. Southern hybridization of genomic DNA with a V kappa Ser-specific cDNA probe has demonstrated a single strongly hybridizing DNA fragment in all strains of mice tested. Characteristic restriction enzyme polymorphisms define the V kappa Ser alleles of expressor (Igk-VSera) and nonexpressor (Igk-VSerb) strains. In the present study, the unrearranged V kappa Ser gene and its flanking regions from an expressor (C.C58) and nonexpressor (BALB/c) strain have been cloned and their nucleotide sequences determined. The C.C58 V kappa Ser gene isolated (the Igk-VSera allele) was shown to code for the two phenotypic markers described. While the nucleotide sequence of the BALB/c coding region (the Igk-VSerb allele) shows 97% identity with the C.C58 gene, single nucleotide substitutions lead to structural changes in the encoded protein which render it IB-negative and Ef1a-negative. These differences alone can explain the failure of strains containing the BALB/c allele to express these kappa-chain phenotypic markers. Also, the BALB/c gene contains a single substitution in a conserved octamer sequence approximately equal to 100 nucleotides upstream of the coding region, which could affect its expression. Finally, the C.C58 allele contains a BAM5/R repetitive DNA element approximately equal to 1200 nucleotides upstream of the coding regions that is not present in BALB/c. This element gives rise to the EcoRI and BamHI restriction enzyme polymorphisms, which distinguish the Igk-VSera and Igk-VSerb alleles.

Genetic polymorphism at the mouse immunoglobulin J kappa locus (Igk-J) as demonstrated by Southern hybridization and nucleotide sequence analysis.

Comparison of the nucleotide sequences of the C.C58 M75 myeloma kappa chain gene and the BALB/c germ-line J kappa segments suggested that the J kappa regions of C.C58 and BALB/c might be distinguished by restriction enzyme polymorphisms. This was shown to be the case in Southern hybridizations of Hinf I and Acc I digests of liver DNA from these and other strains with a J kappa-specific probe. Tests of a wide variety of inbred, congenic, recombinant, and recombinant-inbred strains provided evidence for three alleles, Igk-Ja, Igk-Jb, and Igk-Jc, the type strains for which are C58/J, BALB/c, and SJL/J, respectively. Analysis of the B6.PL(85NS) congenic strain suggests that the Igk-J locus lies in the neighborhood of the Lyt-2/Lyt-3 loci, approximately 0.30 cM from the V gene segment determining the Igk-VSer and Igk-Efl polymorphisms. Finally, nucleotide substitutions lead to amino acid sequence differences between the C.C58 M 75 kappa gene and the BALB/c germ line in J kappa 2 and J kappa 4. Two of these substitutions reflect true germ-line differences, raising the possibility that idiotype differences observed among strains could reflect J kappa as well as V kappa differences.

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis.

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full-length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain-specific expression of V kappa Ser-associated phenotypic markers are discussed.