Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.     

Skewed Th1/Th2 immune response to Sarcoptes scabiei

Scabies is a contagious skin disease of humans and many other species of mammals. Previous studies suggested that the balance between the Th1 and Th2 immune responses may influence the outcome of a scabies infestation in a sensitized host. Therefore, in this study, we examined the T-helper cell cytokine profiles of splenocytes and lymph node cells in BALB/c mice that were immunized with scabies extract (primary response), infested with scabies mites (primary response), or immunized and then infested (secondary response). Lymphocyte cytokine expression was analyzed by flow cytometry after staining for intracellular cytokines. Immunization with scabies extract induced production of interferon-gamma (IFNgamma) (Th1 response) by both spleen and lymph node cells. Mice that were infested with scabies increased production of interleukin-4 by lymph node cells and of IFNgamma by splenocytes. Mice that were first immunized and then infested with mites increased production of IFNgamma by both spleen and lymph node cells. However, this increased level of IFNgamma was only about half of that induced by immunization alone. These results suggest that live scabies mites produced something that inhibited IFNgamma production in the lymph nodes of scabies-immunized mice. Our data also indicate that lymphocytes in the spleen and lymph nodes can present different cytokine response profiles.     

Impact of Nef-mediated downregulation of major histocompatibility complex class I on immune response to simian immunodeficiency virus

Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activation. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant forms of SIV exhibited higher levels of CD8(+) T-cell responses 4 to 16 weeks postinfection than seen in monkeys infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to 32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to downregulate MHC class I contributes importantly to the totality of nef function.     

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8)

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.     

Ultra-fast evaluation of protein energies directly from sequence

The structure, function, stability, and many other properties of a protein in a fixed environment are fully specified by its sequence, but in a manner that is difficult to discern. We present a general approach for rapidly mapping sequences directly to their energies on a pre-specified rigid backbone, an important sub-problem in computational protein design and in some methods for protein structure prediction. The cluster expansion (CE) method that we employ can, in principle, be extended to model any computable or measurable protein property directly as a function of sequence. Here we show how CE can be applied to the problem of computational protein design, and use it to derive excellent approximations of physical potentials. The approach provides several attractive advantages. First, following a one-time derivation of a CE expansion, the amount of time necessary to evaluate the energy of a sequence adopting a specified backbone conformation is reduced by a factor of 10(7) compared to standard full-atom methods for the same task. Second, the agreement between two full-atom methods that we tested and their CE sequence-based expressions is very high (root mean square deviation 1.1-4.7 kcal/mol, R2 = 0.7-1.0). Third, the functional form of the CE energy expression is such that individual terms of the expansion have clear physical interpretations. We derived expressions for the energies of three classic protein design targets-a coiled coil, a zinc finger, and a WW domain-as functions of sequence, and examined the most significant terms. Single-residue and residue-pair interactions are sufficient to accurately capture the energetics of the dimeric coiled coil, whereas higher-order contributions are important for the two more globular folds. For the task of designing novel zinc-finger sequences, a CE-derived energy function provides significantly better solutions than a standard design protocol, in comparable computation time. Given these advantages, CE is likely to find many uses in computational structural modeling.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Quantitative genomics of aggressive behavior in Drosophila melanogaster

Aggressive behavior is important for animal survival and reproduction, and excessive aggression is an enormous social and economic burden for human society. Although the role of biogenic amines in modulating aggressive behavior is well characterized, other genetic mechanisms affecting this complex behavior remain elusive. Here, we developed an assay to rapidly quantify aggressive behavior in Drosophila melanogaster, and generated replicate selection lines with divergent levels of aggression. The realized heritability of aggressive behavior was approximately 0.10, and the phenotypic response to selection specifically affected aggression. We used whole-genome expression analysis to identify 1,539 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. We quantified the aggressive behavior of 19 mutations in candidate genes that were generated in a common co-isogenic background, and identified 15 novel genes affecting aggressive behavior. Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex traits.     

Chronic intermittent hypoxia alters NE reactivity and mechanics of skeletal muscle resistance arteries.

Although arterial dilator reactivity is severely impaired during exposure of animals to chronic intermittent hypoxia (CIH), few studies have characterized vasoconstrictor responsiveness in resistance arteries of this model of sleep-disordered breathing. Sprague-Dawley rats were exposed to CIH (10% inspired O2 fraction for 1 min at 4-min intervals; 12 h/day) for 14 days. Control rats were housed under normoxic conditions. Diameters of isolated gracilis muscle resistance arteries (GA; 120-150 microm) were measured by television microscopy before and during exposure to norepinephrine (NE) and angiotensin II (ANG II) and at various intraluminal pressures between 20 and 140 mmHg in normal and Ca2+-free physiological salt solution. There was no difference in the ability of GA to constrict in response to ANG II (P = 0.42; not significant; 10(-10)-10(-7) M). However, resting tone, myogenic activation, and vasoconstrictor responses to NE (P < 0.001; 10(-9)-10(-6) M) were reduced in CIH vs. controls. Treatment of rats with the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol; 1 mM) in the drinking water restored myogenic responses and NE-induced constrictions of CIH rats, suggesting that elevated superoxide production during exposure to CIH attenuates vasoconstrictor responsiveness to NE and myogenic activation in skeletal muscle resistance arteries. CIH also leads to an increased stiffness and reduced vessel wall distensibility that were not correctable with oral tempol treatment.     

Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain

In the current protocol, we describe the Congo red staining method and a method for separately quantifying vascular and parenchymal amyloid deposits in brain tissue sections. Congo red staining detects amyloid deposits in brain tissue of amyloid precursor protein transgenic mice and human Alzheimer's tissue. It detects compacted amyloid in a beta-sheet secondary structure and labels amyloid in both the brain parenchyma (amyloid plaques) and blood vessels. Congophilic amyloid in blood vessels is called cerebral amyloid angiopathy (CAA). To date, analysis of CAA has largely used a severity rating scale, including both qualitative and quantitative characteristics. Here, we describe a simple method for quantifying total Congophilic staining and resolution of this staining into the parenchymal and vascular components based on morphological criteria. It is becoming increasingly important to separately quantify various components of the Alzheimer's pathology, given the advancement of amyloid-lowering therapies into clinical trials. The entire procedure for the Congo red staining can be performed at room temperature (20-25 degrees C) in a fume hood. The staining protocol should take 1 h 30 min including time for coverslipping slides. Time required for image analysis depends greatly on the number of samples being analyzed and the software being used. In our hands, 30 images can be collected per hour and quantified in a further 2 h.