Daniel Goldberg

The present status of 38 couples who had been treated at a clinic for sexual dysfunction 3 years previously was determined by a self-report assessment battery. The battery consisted of the Sexual Interaction Inventory, the Locke-Wallace Marriage Inventory, and the Sexual History Form completed at pretreatment, immediately posttreatment, 3 months after treatment, and at 3-year follow-up. An additional Follow-up Questionnaire was completed at the 3-year point only. At 3-year follow-up, analysis of data by diagnostic category indicated that sexual desire dysfunction for both men and women was particularly resistant to sustained behavioral change. Men with erectile difficulty reported significant improvement in their ability to maintain erections during intercourse but not in their ability to achieve erections prior to intercourse. Data from men with premature ejaculation revealed some immediate significant posttherapy gains, which, with the exception of length of foreplay, were not sustained at 3-year follow-up. For women with global inorgasmia, a significant increase in orgasmic response was reported. Data from women with situational orgasmic difficulties indicated some success in improving frequency of orgasm through masturbation and through genital caress; however, these changes did not reach statistical significance. Across all diagnostic categories, both men and women respondents reported increased satisfaction in their sexual relationship. Satisfaction in the marital relationship showed a more varied response pattern.

A number of single-digit nanomolar, low-molecular-weight plasmepsin II aspartyl protease inhibitors have been identified using combinatorial chemistry and structure-based design. By identifying multiple, small-molecule inhibitors using the parallel synthesis of several focused libraries, it was possible to select for compounds with desirable characteristics including enzyme specificity and minimal binding to serum proteins. The best inhibitors identified have Ki's of 2-10 nM, molecular weights between 594 and 650 Da, between 3- and 15-fold selectivity toward plasmepsin II over cathepsin D, the most closely related human protease, good calculated log P values (2.86-4.56), and no apparent binding to human serum albumin at 1 mg/mL in an in vitro assay. These compounds represent the most potent non-peptide plasmepsin II inhibitors reported to date.

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.

Hemoglobin degradation in intraerythrocytic malaria parasites is a vast process that occurs in an acidic digestive vacuole. Proteases that participate in this catabolic pathway have been defined. Studies of protease biosynthesis have revealed unusual targeting and activation mechanisms. Oxygen radicals and heme are released during proteolysis and must be detoxified by dismutation and polymerization, respectively. The quinoline antimalarials appear to act by preventing sequestration of this toxic heme. Understanding the disposition of hemoglobin has allowed identification of essential processes and metabolic weakpoints that can be exploited to combat this scourge of mankind.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.