Evidence of a role for insulin-like growth factor binding protein (IGFBP)-3 in metabolic regulation.

IGF-binding protein (IGFBP)-3 is a metabolic regulator that has been shown to inhibit insulin-stimulated glucose uptake in murine models. This finding contrasts with epidemiological evidence of decreased serum IGFBP-3 in patients with type 2 diabetes. The purpose of this study was to clarify the role of IGFBP-3 in metabolism. Four-week-old male IGFBP-3(-/-) and control mice were subjected to a high-fat diet (HFD) for 12 wk. IGFBP-3(-/-) mice were heavier before the initiation of HFD and at the end of the study period. Resting metabolic rate was significantly decreased in knockout mice; however, respiratory exchange ratio was not significantly different. Fasting blood glucose and insulin levels were significantly elevated in IGFBP-3(-/-) mice. However, IGFBP-3(-/-) mice had relatively normal glucose tolerance because the relative glucose excursion over time was not different between the groups. During hyperinsulinemic clamps, IGFBP-3(-/-) mice had increased basal hepatic glucose production, but after insulin stimulation, no differences in hepatic glucose production were observed. A second cohort of older IGFBP-3(-/-) mice on HFD displayed unexpected evidence of hepatic steatosis. In summary, glucose tolerance and clamp testing indicate that IGFBP-3(-/-) mice preserve insulin sensitivity despite evidence of increased basal glucose turnover and hepatic steatosis. We provide evidence that genetic deletion of IGFBP-3 modulates hepatic carbohydrate and lipid metabolism.

Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%; the similarity was lower than 85% Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency.

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

A method for the simulation of normal, carrier and affected controls for PCR-RFLP screening of a genetic disease in dairy cattle.

A technique is described that may be used to create in vitro mutations in PCR templates to generate affected and carrier controls for diagnostic testing when DNA from such individuals is not easily obtained. The method is demonstrated for a PCR-RFLP diagnostic test of the genetic disorder BLAD (Bovine Leukocyte Adhesion Deficiency).

Marrow versus infection in the Charcot joint: indium-111 leukocyte and technetium-99m sulfur colloid scintigraphy.

UNLABELLED: This study evaluated the role of combined leukocyte/marrow scintigraphy in the assessment of the neuropathic or Charcot joint. METHODS: Seventeen patients with (111)In-labeled leukocyte accumulation in 20 radiographically confirmed Charcot joints underwent 99mTc-sulfur colloid marrow scintigraphy. Studies demonstrating labeled leukocyte accumulation without corresponding activity on marrow images were classified as positive for osteomyelitis. Six of the patients also underwent three-phase bone scintigraphy. Bone scans were interpreted as positive for osteomyelitis when focal hyperperfusion, focal hyperemia and focal bony uptake on delayed images were present. Bone images were also interpreted together with labeled leukocyte images using two different criteria for a positive study. One criterion was the presence of labeled leukocyte activity in a region demonstrating abnormal activity on the bone scan, which was more intense than adjacent marrow activity or marrow activity in the corresponding region of the contralateral foot. The second criterion was either a spatially incongruent distribution of the two tracers or hyperintense activity on the leukocyte study, as compared to the bone scan. RESULTS: Leukocyte/marrow studies were positive for osteomyelitis in 4 of the 20 neuropathic joints. Osteomyelitis was present in three of the four joints, whereas in the fourth, infection was confined to overlying soft tissues. None of the 16 neuropathic joints with negative leukocyte/marrow scans were infected. In one patient who underwent below-the-knee amputation, histological analysis confirmed the presence of hematopoietically active marrow corresponding to areas of congruent activity on the leukocyte and marrow images. Three-phase bone scintigraphy was positive in all six neuropathic joints studied; osteomyelitis was present in two of them. Using the first criterion, leukocyte/bone imaging was also positive in all six. Using the second criterion, leukocyte/bone imaging was positive in the two infected neuropathic joints, as well as in three uninfected ones. Leukocyte/marrow scintigraphy was positive in both infected joints and negative in the four without infection. CONCLUSION: Labeled leukocyte accumulation in the uninfected Charcot joint does occur and is related, at least in part, to hematopoietically active marrow. Leukocyte/marrow scintigraphy is a reliable way to differentiate between marrow and infection as the cause of labeled leukocyte accumulation in the neuropathic joint and, in this series, was superior to both three-phase bone scintigraphy and combined leukocyte/bone scintigraphy.