Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency

Homocysteine can be methylated to form methionine by the cobalamin- (Cbl) and folate-dependent enzyme, methionine synthase; serum levels of total homocysteine are elevated in greater than 95% of patients with either Cbl or folate deficiency. Homocysteine can also condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine beta-synthase. Cystathionine is subsequently cleaved to cysteine and alpha-ketobutyrate by the pyridoxal phosphate-dependent enzyme gamma-cystathionase. To assess levels of cystathionine in Cbl and folate deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of these vitamins. The normal range for serum cystathionine was 65 to 301 nmol/L (median = 126 nmol/L) for 50 normal blood donors. In 30 patients with clinically confirmed Cbl deficiency, values for cystathionine ranged from 208 nmol/L to 2,920 nmol/L (median = 816 nmol/L) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 nmol/L to 4,150 nmol/L (median = 1,560 nmol/L) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine beta-synthase deficiency had high values for serum-total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 nmol/L to 114 nmol/L even though they were on treatment with pyridoxine and had partially responded. One patient with a defect in the synthesis of 5-CH3- tetrahydrofolate and five patients with defects in the synthesis of CH3- Cbl had high values for serum-total homocysteine and high values for cystathionine that ranged from 311 nmol/L to 1,500 nmol/L even though they were on treatment with folic acid and Cbl, respectively, and had partially responded. We conclude that levels of cystathionine are evaluated in the serum of most patients with Cbl and folate deficiency and that they are useful in the differential diagnosis of an elevated serum-total homocysteine level.     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.     

Biological evidence supports an early and complex emergence of the Isthmus of Panama

The linking of North and South America by the Isthmus of Panama had major impacts on global climate, oceanic and atmospheric currents, and biodiversity, yet the timing of this critical event remains contentious. The Isthmus is traditionally understood to have fully closed by ca. 3.5 million years ago (Ma), and this date has been used as a benchmark for oceanographic, climatic, and evolutionary research, but recent evidence suggests a more complex geological formation. Here, we analyze both molecular and fossil data to evaluate the tempo of biotic exchange across the Americas in light of geological evidence. We demonstrate significant waves of dispersal of terrestrial organisms at approximately ca. 20 and 6 Ma and corresponding events separating marine organisms in the Atlantic and Pacific oceans at ca. 23 and 7 Ma. The direction of dispersal and their rates were symmetrical until the last ca. 6 Ma, when northern migration of South American lineages increased significantly. Variability among taxa in their timing of dispersal or vicariance across the Isthmus is not explained by the ecological factors tested in these analyses, including biome type, dispersal ability, and elevation preference. Migration was therefore not generally regulated by intrinsic traits but more likely reflects the presence of emergent terrain several millions of years earlier than commonly assumed. These results indicate that the dramatic biotic turnover associated with the Great American Biotic Interchange was a long and complex process that began as early as the Oligocene–Miocene transition.The Isthmus of Panama is the narrow strip of land that connects North and South America and divides the Atlantic and Pacific oceans. The emergence of the Isthmus initiated one of the largest episodes of biological migration between previously disconnected landmasses, the Great American Biotic Interchange (GABI) (1–5), one of the best natural experiments on invasive species. The closure of the Central American Seaway (CAS, the oceanic pathway along the tectonic boundary between South America and the Panama Block) and rise of the Isthmus have been linked to the onset of both thermohaline oceanic circulation and northern hemisphere glaciation (6–8). Despite its broad importance, the formation of the Isthmus and its impact on the rich biodiversity of the Americas remains contentious (9). Therefore, a better understanding of when the formation of the Isthmus of Panama occurred has important implications in several scientific fields across multiple disciplines.The timing of Isthmus formation has been assessed through different proxies. Previous studies have long suggested full closure by 3.5 Ma (7, 8, 10–16). More recent geological work has suggested a longer and more complex formation, where the initial collision between South America and the Panama Block occurred between 25 and 23 Ma (17). By 20 Ma the Panama Block is suggested to have been connected to North America (18–21) and the width of the CAS to be 200 km (19, 20). Full closure of the CAS occurred by 10 Ma, ending the exchange of deep and intermediate waters between the Caribbean and the Pacific (11, 19, 20, 22–24). However, the exchange of shallow waters between these oceans likely continued along pathways other than the CAS for many millions of years (7, 8, 10–16, 25).Over the past two decades, hundreds of studies have assumed the Isthmus of Panama to have closed at 3.5 Ma (SI Appendix), causing the separation of widespread marine populations into distinct Pacific and Caribbean groups (vicariance) and the first possible dispersals between North and South America (with the exception of stochastic long-distance dispersals). Here, we address the following questions: Given the complexities and the recent evidence of a much older geological history of the Isthmus of Panama, is 3.5 Ma an adequate age for those events? Were the suggested water corridors across the Isthmus—even if shallow and narrow—indeed effective barriers against both dispersal of terrestrial organisms and conduits of marine ones until a full closure at 3.5 Ma? We address these questions using comprehensive biological data from living and fossil organisms, where the assumption is that any well-developed terrestrial corridor would lead to both more frequent biotic dispersal between North and South America as well as a division of widespread marine organisms into distinct Caribbean and Pacific lineages (26). Biological data provide a powerful tool for this purpose compared with geological evidence, which cannot inform on the subtle differences between exposed land and shallow waters that can be crucial to the dispersal of organisms.Recent attempts to synthesize dispersal patterns across the Isthmus of Panama are based on dated phylogenies (27–29) that have included data that used a 3.5 Ma Isthmus closure as a calibration point (e.g., in calculating mutation rate). These assumptions lead to circularity if the goal is to examine timing of dispersal or vicariance across the Isthmus. Our molecular analyses differ from previous studies both qualitatively (by the elimination of the timing of the emergence of the Isthmus of Panama and the contrast between patterns in marine and terrestrial taxa) and quantitatively (735% more data points than a previous cross-taxonomic analysis) (27) and serve as a comparison with a comprehensive fossil dataset.Another crucial aspect of the formation of the Isthmus of Panama and its impact on biological diversity entails the direction of terrestrial dispersal (e.g., north to south) and the ecology of dispersing organisms. In his seminal work on the GABI, Simpson (1) used the mammal fossil record to suggest that North American taxa had a competitive advantage over the South American fauna in that more North American taxa dispersed, survived, and diversified in southern ranges. Recent studies have suggested that ecological barriers to dispersal, such as dry savanna-like environments (30) or reduced rain forest cover (31), also prevented tropical South American taxa from migrating successfully to the north.To explicitly address the timing of the formation of the Isthmus of Panama and its effect on geographical distributions of biota, we develop a Migration Rates Through Time (MRTT) model. Using this approach, in which migration is defined as dispersal and vicariance events collectively, we conduct an analysis of over 400 data points (SI Appendix, 1.2) of molecular divergence dates conferring dispersal events between North and South America or vicariance between the eastern Pacific and the Caribbean Sea. We compare results from extant data with over 23,000 records of American fossil mammals. Our results corroborate recent geological evidence that rather than a Pliocene, time-limited, single event, the formation of the Isthmus of Panama and the GABI were long and complex processes that began as early as the Oligocene–Miocene transition. We also show incongruent patterns in the direction of migration when comparing the molecular and fossil data, possibly reflecting differential diversification rates following dispersal. None of the ecological factors measured in the analyses can explain the statistical differences in the timing of migration across the Isthmus region among different taxa, and together with the MRTT results, this suggests that rather than intrinsic (biological) traits, extrinsic factors such as the presence of emergent land likely drove these patterns.     

Periodontal status of HIV infected patients with special reference to CD4 cell count in West Bengal,India

ObjectiveTo evaluate the periodontal status of HIV seropositive patients and to find out if any correlation exists between the severity of periodontal disease and the CD4 cell count in HIV patients.MethodsOne hundred and thirty patients attending the Viral Diseases OPD, Calcutta School of Tropical Medicine, Kolkata were examined. They were grouped according to the CD4 cell count as Group A – Subjects with CD4 Cell count < 200/μL and Group B – Subjects with CD4 Cell count≥200/μL. Their community periodontal index of treatment needs (CPITN) score were recorded.ResultsIt was found that most of the patients in each group were having score ‘2’ (i.e. presence of supra or subgingival calculus), as their highest score. A statistically significant association was found between immune status as depicted by CD4 cell count and periodontal status as shown by highest CPITN score in the present study.ConclusionsThe present study confirms the effect of immunosuppression on periodontal diseases in HIV infected patients.     

Correlation of P-glycoprotein expression and function in childhood acute leukemia: a children's cancer group study

Clinical drug resistance may be attributed to the simultaneous selection and expression of genes modulating the uptake and metabolism of chemotherapeutic agents. P-glycoprotein (P-gp) functions as a membrane-associated drug efflux pump whose increased expression results in resistance to anthracyclines, epipodophyllotoxins, vinca alkaloids, and some alkylating agents. This type of resistance occurs as both de novo and acquired resistance to therapy for leukemia. We have studied P- gp expression and function in childhood acute leukemias by developing a series of doxorubicin- and vincristine-selected CEM, T-cell lymphoblastoid cell lines that recapitulate the low levels of expression and resistance seen clinically. These cell lines have been used to develop flow cytometric assays for the semiquantitative measurements of P-gp expression with the MRK16 monoclonal antibody and P-gp function using the enhanced retention of rhodamine 123 in the presence of verapamil, a resistance modulator. Kolmogorov-Smirnov statistics, represented by the D measurement, are used to determine the difference in level of P-gp expression by comparing MRK16 staining to an IgG2a isotype control. When D is > 0.09, there is an excellent correlation (R = 0.82) between P-gp expression and function. The evaluation of 107 bone marrow specimens from 84 children with lymphoblastic or myelogenous leukemia showed a statistically significant (P = .004) increase in P-gp function at relapse. P-gp expression at relapse, however, approached but did not reach a significant level (P = .097). Using this methodology, we can identify patients with levels of P-gp expression and function that we can define clinically, as well as children with discordant multidrug resistance phenotypes. This study supports the role of P-gp-mediated drug resistance in childhood leukemia and confirms that P-gp expression and function are measurable in their leukemic blasts. These assays provide the means for the in vitro testing of resistance modulators and the monitoring of in vivo response to treatment with these agents.     

Characterization of the IgG-Fc receptor on human platelets

To determine quantitatively the number and avidity of receptors for the Fc portion of IgG on human platelets, we have measured the binding to platelets of human monomeric monoclonal IgG, and of small covalently crosslinked polymers of IgG1 labeled with 125I. The binding of labeled IgG1 monomers to platelets is too weak to permit quantitation. The binding of dimers or larger polymers of IgG1 is much more avid (greater at 4 degrees C than 37 degrees C), is readily reversible, and is saturable. The number of receptor sites ranges from 400 to 2000 per platelet and the mean equilibrium association constant (Ka) for the binding of dimers at 4 degrees C is 2.2 x 10(7) M-1 +/- 0.9 x 10(7) M- 1. The binding is specific for the Fc portion of IgG, and IgG1 and IgG3 bind to the receptors much more avidly than IgG2 or IgG4. Unlabeled IgG1 dimers are about 7--8-fold more potent in inhibiting binding than are IgG1 monomers, and larger polymers are even more potent than dimers. Thus, the Fc receptors on platelets bind human IgG1 with the same specificity and similar avidity as Fc receptors on polymorphonuclear leukocytes (PMNs), but PMNs have about 300-fold more receptors per unit of surface area than platelets.     

Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria

The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable.     

The response of red cell hexose monophosphate shunt after sulfhydryl inhibition

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydryl blockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.