Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.     

Immunologic Memory Induced by a Glycoconjugate Vaccine in a Murine Adoptive Lymphocyte Transfer Model

We have developed an adoptive cell transfer model in mice to study the ability of a glycoprotein conjugate vaccine to induce immunologic memory for the polysaccharide moiety. We used type III capsular polysaccharide from the clinically relevant pathogen group B streptococci conjugated to tetanus toxoid (GBSIII-TT) as our model vaccine. GBS are a major cause of neonatal infections in humans, and type-specific antibodies to the capsular polysaccharide protect against invasive disease. Adoptive transfer of splenocytes from mice immunized with the GBSIII-TT conjugate vaccine conferred anti-polysaccharide immunologic memory to naive recipient mice. The transfer of memory occurred in a dose-dependent manner. The observed anamnestic immune response was characterized by (i) more rapid kinetics, (ii) isotype switching from immunoglobulin M (IgM) to IgG, and (iii) 10-fold-higher levels of type III-specific IgG antibody than for the primary response in animals with cells transferred from placebo-immunized mice. The adoptive cell transfer model described in this paper can be used for at least two purposes: (i) to evaluate conjugate vaccines with different physicochemical properties for their ability to induce immunologic memory and (ii) to study the cellular interactions required for an immune response to these molecules.     

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera.

Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study.     

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man

Objective　To detect new mutations among 29 glucose-6-phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province. Methods　The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals. Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing. Results　Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified. Nine cases remained to be defined. The G1381A mutation is a novel mis-sense mutation, with a substitution of threonine for alanine (A461T). The resultant G6PD had reduced enzymatic activity. In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation. Conclusion　A novel mis-sense mutation G1381A was found. This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity. The loss of the Stu I restriction site offers a rapid method for the detection of this mutation.     

Passive-aggressive personality disorder: the demise of a syndrome

This article presents an explanation and critique of the rationale for dropping passive-aggressive personality disorder (PAPD) from DSM-IV. The clinical and research literature on PAPD is reviewed along with the historical changes in definition, diagnostic criteria, and usage. PAPD can be reliably diagnosed, is fairly prevalent, and has good internal consistency. Because PAPD is no less valid than other personality disorders, and describes clinical phenomena that are unique among personality disorders, we recommend the reinstatement of PAPD in the official diagnostic nomenclature.     

LIF: not just a leukemia inhibitory factor

Increasingly it seems that many cytokines are pleiotropic, and individual molecules may have critical roles in several different organ systems. LIF exemplifies this phenomenon: it influences embryogenesis, bone and lipid metabolism, and hematopoietic and nervous system function. Many of LIF's effects are reminiscent of those of IL-1, TNF, and TGF-beta. Further, even within a single system, LIF can display totally different effects, i.e. induction of differentiation of one leukemic cell line vs. stimulation of proliferation of another. The corollary to these observations is that there appears to be many parallels in developmental systems. For instance, in the case of neuronal "lineage commitment," the events that relate to migration of neural crest cells along various pathways and their ultimate arrest in different locales demonstrate sufficient analogies to hematopoietic lineage commitment phenomena that, in a provocative review, Anderson coined the term "neuropoiesis". This type of analogy becomes even more intriguing when one realizes that some of the same molecules are regulating neuronal and hematopoietic "lineage" proliferation and differentiation. In this respect, several interleukins in addition to LIF are important in neuronal development, and nerve growth factor turns out to also be a hematopoietic regulatory molecule. Similar parallels are enacted in other organ systems as well. The mediation of identical effects by distinct cytokines bound to unique receptors could conceivably be explained by receptor transmodulation or by overlapping signaling sequences. It is nevertheless also unclear how a single cytokine attached to a single receptor can accomplish varied and opposing effects, although divergent intracellular signaling mechanisms could account for some of these phenomena. Yet another enigma relates to how cells from one system can be properly influenced by a pleiotropic molecule such as LIF without significant "cross-effects" on other potentially responsive systems. Cytokine production that is restricted to certain developmental stages, or very localized distribution and spheres of influence within a microenvironment, could be explanatory. The findings of Rathjan and colleagues, i.e. that LIF exists as both a diffusible molecule and as a molecule incorporated into the extracellular matrix, is of special interest in relation to the above questions. Indeed, the distinctions between the roles of diffusible and immobilized signaling molecules could be crucial to the multiplicity of LIF's actions. Diffusible regulatory factors allow communication between spatially separated cells. Cellular responsiveness to such factors is dictated by the presence of appropriate receptors and postreceptor machinery.(ABSTRACT TRUNCATED AT 400 WORDS)